Methods and devices for ultrasensitive direct detection of microorganisms

ABSTRACT

The present disclosure generally relates to the field of ultrasensitive microbial pathogen detection and identification utilizing genomic sequence recognition.

RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser. No. 16/848,239, filed Apr. 14, 2020, entitled “METHODS AND DEVICES FOR ULTRASENSITIVE DIRECT DETECTION OF MICROORGANISMS”, which claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 62/834,729, filed Apr. 16, 2019, entitled “METHODS AND DEVICES FOR ULTRASENSITIVE DIRECT DETECTION OF MICROORGANISMS”, each of which is incorporated herein by reference in its entirety.

GOVERNMENT SUPPORT

This invention was made with Government support under Grant No. AI124726 awarded by the National Institutes of Health. The Government has certain rights in the invention.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing (H091670002US03-SEQ-CRS.xml; Size: 1,793,901 bytes; and Date of Creation: Jun. 1, 2023) is herein incorporated by reference in its entirety.

TECHNICAL FIELD

The present invention generally relates to the field of microbial pathogen detection and identification utilizing genomic sequence recognition.

BACKGROUND

Molecular assays present unique opportunities for direct detection of microorganisms. However, in blood, they are readily confounded by an overwhelming background of human DNA (hDNA), which limits sample volumes and presents significant problems when microbial loads are low. Methods for the detection of extremely low microbial loads, for 30 example less than 10 cells/ml of specimen, have not been demonstrated to achieve this task accurately and reproducibly. Indeed, in multiple infectious diseases early and accurate detection of the etiologic pathogen may require detection capabilities as low as 1 cell/ml, and perhaps lower. One such infectious disease, exemplified in this disclosure, is Lyme disease.

Lyme disease (LD) is the most prevalent tick-borne disease in North America s and increasingly common in Europe and Asia. Borrelia burgdorferi is the primary causative agent of LD in North America (˜300,000 cases annually), where B. afzelii and B. garinii are common in Europe with ˜90,000 cases annually. As its prevalence and our understanding of the disease grows, recent cases have emerged caused by an additional twelve species. Importantly, there is evidence that disease manifestation, progression, and severity are species-related, underscoring the need for early detection and (preferably) Borrelia-species ID with broad coverage.

Serological methods (gold standard) are limited as they lack sensitivity (antibodies require weeks to reach the required titers) and specificity (due to differential protein expression) and only detect under 20% of cases of early LD. Despite their poor predictive value, these tests are utilized 3.4 million times annually just in the US. Alternatively, blood cultures are non-starters, requiring weeks to yield results given Borrelia's doubling time (12-18 h). For this reason, cultures are not part of a LD workup.

Molecular methods to date for the direct-detection of Borrelia suffer from insufficient clinical sensitivity, largely due to the low microbial loads evident in blood in the early stages of an infection. While improvements in analytical sensitivity via standard approaches (genes, primers, etc.) have improved clinical performance, they are not sufficient to justify routine usage and as such no molecular test has been cleared by the FDA.

It is widely believed that the key limitation with existing molecular assays is that of sampling; blood inputs tested today are far too low. Even external to molecular assays and while not suitable for routine clinical work, the culturing of ˜1 ml blood yielded sensitivities of ˜5-20%, where 9 ml blood cultures yielded ˜50% sensitivity. While Borrelia cultures have notoriously poor recovery, these results are telling as improved sampling yielded significantly higher sensitivity. Indeed, in a study conducted by Wormser and coworkers utilizing small aliquots of cultures seeded by 9 ml blood, though still ‘visually’ negative, qPCR yielded positive results in >70% of early LD cases, underscoring both the importance of blood sampling volumes and the limitations of culture.

Not to be undone, molecular diagnostics have shown results in line with those of culture. LDTs, which typically assay 0.05-0.2 ml of blood demonstrate analytical sensitivities in the range of 10²-10³ cells/ml; resulting in clinical sensitivities of ˜10-20%. In light of this, recent studies have shown that the sampling of larger volumes (1.25-1.75 ml) of blood improves both analytical sensitivity (to 20-100 cells/ml) and clinical sensitivity (up to 40%). Unfortunately, due to sample-preparation limitations only a fraction (33-50%) of this input is available in any single amplification reaction. The probability of pathogens reaching amplification is the sensitivity bottleneck. Thus, while clearly an improvement, the still insufficient clinical sensitivity of these efforts suggests that an even lower LoD is required perhaps even as low as 1-10 cells/ml of blood. The end result is that no direct-detection method of early LD diagnosis is available clinically.

SUMMARY

The present disclosure generally relates to the field of microorganisms, e.g., microbial pathogens, detection and identification utilizing genomic sequence recognition. In particular, the claimed methods, compositions, and kits provide for the ultrasensitive and direct-detection, identification and evaluation of microorganisms present at low levels, e.g., a microbial load below 10 cells/ml, in a sample, e.g., in blood. Direct-detection refers to a capability to detect the microorganism directly in a sample without the need for culturing the sample. Other advantages and novel features of the methods, devices, and kits described herein will become apparent from the following detailed description of various non-limiting embodiments when considered in conjunction with the accompanying figures. In cases where the present specification and a document incorporated by reference include conflicting and/or inconsistent disclosure, the present specification shall control.

In one aspect, disclosed herein is an ultrasensitive method of detecting one or more species of microbial cells in a sample. The method comprises providing a biological sample, wherein the sample is ≥5 ml; selectively lysing the mammalian cells in the sample, including those which contain eukaryotic DNA; separating eukaryotic DNA from the sample by centrifugation; isolating a plurality of microbial genetic materials from the microbial cells; amplifying the plurality of microbial genetic materials; contacting the amplified microbial genetic materials with a plurality of DNA Invading Artificial Nucleic Acids (DIANAs), wherein the plurality of DIANAs comprise one or more sequences that are complementary to a genomic or plasmid sequence of a microbial species; and detecting binding of one or more of the plurality of DIANAs to the microbial genetic material of its respective microbial species, wherein the detection of binding indicates the presence of one or more microbial species in the sample.

In another aspect, disclosed herein is an ultrasensitive method of detecting one or more species of microbial cells in a sample. The method comprises providing a biological sample, wherein the sample is ≥5 ml; selectively lysing the mammalian cells in the sample, including those which contain eukaryotic DNA; separating eukaryotic DNA from the sample by centrifugation; isolating a plurality of microbial genetic materials from the microbial cells; amplifying the plurality of microbial genetic materials; and detecting the amplified microbial genetic material.

In some embodiments, detecting the amplified microbial genetic material comprises: contacting the amplified microbial genetic materials with a plurality of DNA Invading Artificial Nucleic Acids (DIANAs), wherein the plurality of DIANAs comprise one or more sequences that are complementary to a genomic or plasmid sequence of a microbial species; and detecting binding of one or more of the plurality of DIANAs to the microbial genetic material of its respective microbial species, wherein the detection of binding indicates the presence of one or more microbial species in the sample.

In another aspect, disclosed herein is an ultrasensitive method of detecting one or more species of microbial cells in a sample. The method comprises providing a biological sample, wherein the sample is ≥5 ml; selectively lysing the mammalian cells in the sample, including those which contain eukaryotic DNA; separating eukaryotic DNA from the sample by way of capturing and removing the eukaryotic DNA via an anion-exchanger; lysing and thereafter isolating a plurality of microbial genetic materials from the microbial cells; amplifying the plurality of microbial genetic materials; contacting the amplified microbial genetic materials with a plurality of DNA Invading Artificial Nucleic Acids (DIANAs), wherein the plurality of DIANAs comprise one or more sequences that are complementary to a genomic or plasmid sequence of a microbial species; and detecting binding of one or more of the plurality of DIANAs to the microbial genetic material of its respective microbial species, wherein the detection of binding indicates the presence of one or more microbial species in the sample.

In another aspect, disclosed herein is an ultrasensitive method of detecting one or more species of microbial cells in a sample. The method comprises providing a biological sample, wherein the sample is ≥5 ml; selectively lysing the mammalian cells in the sample, including those which contain eukaryotic DNA; separating eukaryotic DNA from the sample by way of capturing and removing the eukaryotic DNA via an anion-exchanger; lysing and thereafter isolating a plurality of microbial genetic materials from the microbial cells; amplifying the plurality of microbial genetic materials; and detecting the amplified microbial genetic material.

In some embodiments, detecting the amplified microbial genetic material comprises: contacting the amplified microbial genetic materials with a plurality of DNA Invading Artificial Nucleic Acids (DIANAs), wherein the plurality of DIANAs comprise one or more sequences that are complementary to a genomic or plasmid sequence of a microbial species; and detecting binding of one or more of the plurality of DIANAs to the microbial genetic material of its respective microbial species, wherein the detection of binding indicates the presence of one or more microbial species in the sample.

In another aspect, disclosed herein is a method of identifying one or more species of Borrelia microbial cells in a sample. The method comprises selectively lysing the mammalian cells in a biological sample, including those which contain eukaryotic DNA; depleting eukaryotic DNA from the sample; lysing one or more microbial cells in the sample, wherein the lysing of one or more microbial cells releases a plurality of microbial genetic materials; isolating the plurality of microbial genetic materials from the sample; amplifying the plurality of microbial genetic materials; contacting the amplified microbial genetic materials with a plurality of DNA Invading Artificial Nucleic Acids (DIANAs), wherein the plurality of DIANAs comprise one or more sequences that are complementary to a genomic or plasmid sequence of a Borrelia species; and detecting binding of one or more of the plurality of DIANAs to the microbial genetic material of its respective Borrelia species, wherein the detection of binding indicates the presence of one or more Borrelia microbial species in the sample.

In another aspect, disclosed herein is a method of identifying one or more species of Borrelia microbial cells in a sample. The method comprises selectively lysing the mammalian cells in a biological sample, including those which contain eukaryotic DNA; depleting eukaryotic DNA from the sample; lysing one or more microbial cells in the sample, wherein the lysing of one or more microbial cells releases a plurality of microbial genetic materials isolating the plurality of microbial genetic materials from the sample; amplifying the plurality of microbial genetic materials; and detecting the amplified microbial genetic material.

In some embodiments, detecting the amplified microbial genetic material comprises contacting the amplified microbial genetic materials with a plurality of DNA Invading Artificial Nucleic Acids (DIANAs), wherein the plurality of DIANAs comprise one or more sequences that are complementary to a genomic or plasmid sequence of a microbial species; and detecting binding of one or more of the plurality of DIANAs to the microbial genetic material of its respective microbial species, wherein the detection of binding indicates the presence of one or more microbial species in the sample.

In some embodiments, the method further comprises separating eukaryotic DNA from the sample by centrifugation prior to lysing one or more microbial cells in the sample.

In another aspect, disclosed herein is a method of identifying one or more species of Borrelia microbial cells in a sample from a subject. The method comprises isolating the plurality of microbial genetic materials from the sample; amplifying the plurality of microbial genetic materials; contacting the amplified microbial genetic materials with a plurality of DNA Invading Artificial Nucleic Acids (DIANAs), wherein the plurality of DIANAs comprise one or more sequences selected from the group consisting of SEQ ID NOs: 1-1358; and detecting binding of one or more of the plurality of DIANAs to the microbial genetic material of its respective Borrelia species, wherein the detection of binding indicates the presence of one or more Borrelia microbial species in the sample.

In some embodiments, the method further comprises (i) selectively lysing the mammalian cells in the sample, including those which contain eukaryotic DNA; and (ii) separating free eukaryotic DNA from the sample by contacting the sample with anionic-exchange microparticles prior to lysing one or more microbial cells in the sample and isolating the plurality of microbial genetic materials from the sample.

In another aspect, disclosed herein is a method of detecting one or more species of microbial cells in a sample. The method comprises providing a biological sample from a subject, wherein the sample is ≥5 ml; selectively lysing the mammalian cells in the sample, including those which contain eukaryotic DNA; separating eukaryotic DNA from the sample by size exclusion chromatography; lysing one or more microbial cells from the sample; isolating a plurality of microbial genetic materials from the sample; amplifying the plurality of microbial genetic materials; contacting the amplified microbial genetic materials with a plurality of DNA Invading Artificial Nucleic Acids (DIANAs), wherein the plurality of DIANAs comprise one or more sequences that are complementary to a genomic or plasmid sequence of a microbial species; and detecting binding of one or more of the plurality of DIANAs to the microbial genetic material of its respective microbial species, wherein the detection of binding indicates the presence of one or more microbial species in the sample.

In any of the foregoing embodiments, removing eukaryotic DNA from the sample by centrifugation comprises, incorporating a plurality of microparticles into the sample; centrifuging the sample; and separating the supernatant containing eukaryotic DNA from the concentrate containing the microparticles and microbial cells. In some embodiments. the plurality of particles comprises one or more of the following: (i) particles having a diameter of approximately 5-8 μM; (ii) particles having a diameter of approximately 1 μM; and (iii) particles having a diameter of approximately 0.2-0.9 μM. In some embodiments, the sample further comprises a control. In some embodiments, the control comprises a live microorganism.

In any of the foregoing embodiments, the method further comprises: lysing one or more microbial cells in the sample prior to isolating a plurality of microbial genetic materials from the sample, wherein the lysing of one or more microbial cells releases the plurality of microbial genetic materials.

In any of the foregoing embodiments, the method is for detecting Borrelia species. In some embodiments, the genomic or plasmid sequence comprises a sequence of a plasmid selected from BB147, cp9, cp26, cp32-1, cp32-3, cp32-4, cp32-6, cp32-7, cp32-8, cp32-9, 1p5, 1p17,1p21,1p25A,1p25B,1p28-1A,1p28-1B,1p28-2,1p28-3,1p28-4,1p36,1p38,1p54, 1p56, or V1sE. In some embodiments, the genomic or plasmid sequence of a Borrelia species comprises a genomic sequence selected from OspA, OspB, OspC, fla, or omp66. In some embodiments, the plurality of DIANAs comprise one or more sequences selected from the group consisting of SEQ ID NOs: 1-1358.

In any of the foregoing embodiments, the microbial load of the sample is less than 50 cells/sample, less than 10 cells/sample, less than 8 cells/sample, less than 6 cells/sample, less than 4 cells/sample, less than 2 cells/sample.

In any of the foregoing embodiments, the microbial load of the sample is less than 100 cells/mL of sample, 10 cells/mL of sample, less than 8 cells/mL of sample, less than 6 cells/mL of sample, less than 4 cells/mL of sample, less than 2 cells/mL of sample.

In any of the foregoing embodiments, the microbial load of the sample is less than 50 CFU/sample, less than 10 CFU/sample, less than 8 CFU/sample, less than 6 CFU/sample, less than 4 CFU/sample, less than 2 CFU/sample.

In any of the foregoing embodiments, the microbial load of the sample is less than 100 CFU/mL of sample, 10 CFU/mL of sample, less than 8 CFU/mL of sample, less than 6 CFU/mL of sample, less than 4 CFU/mL of sample, less than 2 CFU/mL of sample.

In any of the foregoing embodiments, the sample is a blood sample.

In any of the foregoing embodiments, the volume of the sample is 10-20 ml.

In another aspect, disclosed herein is a composition comprising one or more DIANAs comprising a sequence selected from the group consisting of SEQ ID NO. 1-1358. In some embodiments, one or more of the DIANAs comprises at least one LNA, at least one PNA, at least one bis-PNA, at least one pcPNA, at least one, yPNA, or at least one BNA.

In another aspect, disclosed herein is a kit comprising one or more DIANAs, wherein the DIANAs comprise one or more sequences selected from the group consisting of SEQ ID NO. 1-1358. In some embodiments, one or more of the DIANAs comprises at least one LNA, at least one PNA, at least one bis-PNA, at least one pcPNA, at least one, yPNA, or at least one BNA.

In another aspect, disclosed herein is a composition comprising:

a magnesium salt; and a compound of Formula 1:

wherein R₁ is selected from the group consisting of optionally substituted, branched or unbranched, saturated or unsaturated C₁-C₈ aliphatic; optionally substituted, saturated or unsaturated C₃-C₁₄ carbocyclic; optionally substituted, saturated or unsaturated 3-8 membered heterocyclic; optionally substituted, branched or unbranched, saturated or unsaturated ((R_(a))_(q)—(C═O)—(R_(a))_(q))_(p); optionally substituted C₆-C₁₄ aryl; and optionally substituted 3-8 membered heteroaryl; and/or any suitable combinations thereof;

wherein R₂ is selected from the group consisting of hydrogen; optionally substituted, branched or unbranched, saturated or unsaturated C₁-C₂₈ aliphatic; optionally substituted, branched or unbranched, saturated or unsaturated —(R_(b)—(O—R_(b))_(n)—O—R_(b))_(p); optionally substituted, branched or unbranched, saturated or unsaturated —(R_(b)—(O—R_(b))_(n)—NH—R_(b))_(p); optionally substituted, branched or unbranched, saturated or unsaturated —(R_(b)—(O—R_(b)—O)_(n)—S—R_(b))_(p); optionally substituted, branched or unbranched, saturated or unsaturated —(R_(b)—(S—R_(b))_(n)—S—R_(b))_(p); optionally substituted C₆-C₁₄ aryl; optionally substituted 3-8 membered heteroaryl; optionally substituted, saturated or unsaturated C₃-C₁₄ carbocyclic; optionally substituted, saturated or unsaturated 3-8 membered heterocyclic; optionally substituted, branched or unbranched, saturated or unsaturated —(C═O)—(R_(b)); optionally substituted, branched or unbranched, saturated or unsaturated —((R_(a))_(q)—O—(R_(a))_(q))_(p)—; optionally substituted, branched or unbranched, saturated or unsaturated —((R_(a))_(q)—NH—(R_(a))_(q))_(p)—; optionally substituted, branched or unbranched, saturated or unsaturated —((R_(a))_(q)—N(R_(a))—(R_(a))_(q))_(p)—; and optionally substituted, branched or unbranched, saturated or unsaturated —((R_(a))_(q)—S—(R_(a))_(q))_(p)—; and/or any suitable combinations thereof;

wherein each occurrence of R a is independently C₁-C₈ aliphatic or C₆-C₁₄ aryl;

wherein each occurrence of R_(b) is independently C₁-C₁₅ aliphatic or C₆-C₁₄ aryl;

wherein each occurrence of subscript q is independently an integer between 0 and 1, wherein each occurrence of subscript p is independently an integer between 1 and 6, inclusive; and wherein each occurrence of subscript n is independently an integer between 0 and 14, inclusive.

In some embodiments, R₁ is independently selected from the group consisting of optionally substituted, branched or unbranched C₁-C₈ alkyl; optionally substituted, branched or unbranched C₂-C₈ alkenyl; and optionally substituted, branched or unbranched C₂-C₈ alkynyl. In some embodiments, R₁ is optionally substituted, branched or unbranched C₁-C₈ alkyl. In some embodiments, R₁ is C₂ alkyl.

In some embodiments, R₂ is independently selected from the group consisting of optionally substituted, branched or unbranched C₁-C₂₈ alkyl, optionally substituted, branched or unbranched C₂-C₂₈ alkenyl, optionally substituted, branched or unbranched C₂-C₂₄ alkynyl, optionally substituted C₆-C₁₄ aryl, optionally substituted C₃-C₁₄ cycloalkyl, optionally substituted —CH₂—(OCH₂—CH₂)_(n)O—CH₃, optionally substituted —CH₂—(OCH₂—CH₂)_(n)NHCH₃, optionally substituted —CH₂—(OCH₂—CH₂O)_(n)SCH₃, optionally substituted —CH₂—(SCH₂—CH₂)_(n)SCH₃, and optionally substituted —OC—(CH₂)_(n)CH₃. In some embodiments, R₂ is independently selected from the group consisting of optionally substituted, branched or unbranched C₁-C₂₈ alkyl and optionally substituted, branched or unbranched C₂-C₂₈ alkenyl. In some embodiments, R₂ is independently selected from the group consisting of optionally substituted, branched or unbranched C₄-C₁₆ alkyl and C₁₁ alkenyl. In some embodiments, R₂ is C₁₆ alkyl.

In some embodiments, the compound of Formula 1 is selected from the group consisting of:

In some embodiments, the compound of Formula 1 is

In some embodiments, a concentration of the compound of Formula 1 is between 1 mM and 1,000 mM, inclusive. In some embodiments, a concentration of the compound of Formula 1 is between 1 mM and 100 mM, inclusive. In some embodiments, a concentration of the compound of Formula 1 is between 5 mM and 500 mM, inclusive.

In some embodiments, the magnesium salt is selected from the group consisting of MgCl₂, MgCO₃, MgSO₄, and MgBr₂. In some embodiments, a concentration of the magnesium salt is between 1 mM and 100 mM, inclusive. In some embodiments, a concentration of the magnesium salt is between 5 mM and 50 mM, inclusive.

In some embodiments, the composition further comprises a pH between 8 and 11.5, inclusive.

In some embodiments, the composition further comprises blood. In some embodiments, the composition comprises between 20% and 60%, inclusive, of the blood by volume.

In some embodiments, in any of the methods described herein, selectively lysing the mammalian cells in the sample, including those which contain eukaryotic DNA comprises contacting the sample with any of the compositions comprising a compound of Formula 1 described herein.

In another aspect, described herein is an ultrasensitive method of detecting one or more species of microbial cells in a sample, the method comprising: selectively lysing the mammalian cells in a biological sample, including those which contain eukaryotic DNA by contacting the sample with any of the compositions comprising a compound of Formula 1 described herein; and amplifying a plurality of microbial genetic materials in the biological sample; and detecting the amplified microbial genetic material. In some embodiments, the method is for detecting Borrelia.

In some embodiments, detecting the amplified microbial genetic material comprises:

contacting the amplified microbial genetic materials with a plurality of DNA Invading Artificial Nucleic Acids (DIANAs), wherein the plurality of DIANAs comprise one or more sequences that are complementary to a genomic or plasmid sequence of a microbial species; and detecting binding of one or more of the plurality of DIANAs to the microbial genetic material of its respective microbial species, wherein the detection of binding indicates the presence of one or more microbial species in the sample.

In some embodiments, the method further comprises providing a biological sample.

In some embodiments, the method further comprises: (i) separating eukaryotic DNA from the sample by centrifugation; and/or (ii) isolating a plurality of microbial genetic materials from the microbial cells after selectively lysing the mammalian cells in a biological sample.

In some embodiments, the method further comprises: (i) separating free eukaryotic DNA from the sample by contacting the sample with anionic-exchange microparticles; and/or (ii) removing the anionic-exchange microparticle from the sample (iii) isolating a plurality of microbial genetic materials from the microbial cells after selectively lysing the mammalian cells in a biological sample.

In another aspect, described herein is a method of selectively lysing mammalian cells in biological sample comprising mammalian cells, including those which contain eukaryotic DNA, and Borrelia cells, the method comprising contacting the sample with any of the compositions comprising a compound of Formula 1 described herein.

In some embodiments, the comprising a compound of Formula 1 is added to the sample to a final concentration of 0.25 mM and 250 mM, inclusive. In some embodiments, the comprising a compound of Formula 1 is contacted to the sample to a final concentration of 0.5 mM and 100 mM, inclusive. In some embodiments, the comprising a compound of Formula 1 is added to the sample to a final concentration of 1 mM and 50 mM, inclusive. In some embodiments, selectively lysing the mammalians cells further comprises contacting the sample with a magnesium salt selected from the group consisting of MgCl₂, MgCO₃, MgSO₄, and MgBr₂. In some embodiments, the magnesium salt is contacted to the sample to a final concentration of 1 mM and 50 mM, inclusive. In some embodiments, the magnesium salt is contacted to the sample to a final concentration of 5 mM and 25 mM, inclusive.

In some embodiments, selectively lysing the mammalians cells further comprises adjusting the pH of the sample to between 8 and 11.5, inclusive.

In some embodiments, during the selective lysis, the sample comprises between 20% and 60%, inclusive, blood by volume.

BRIEF DESCRIPTION OF THE FIGURES

Non-limiting embodiments of the present invention will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the invention shown where illustration is not necessary to allow those of ordinary skill in the art to understand the invention. In the figures:

FIG. 1 is a schematic diagram of the DIANA invasion process.

FIG. 2 is a schematic diagram of the ultra-sensitive detection methods described herein.

FIG. 3 is a schematic diagram of removal of eukaryotic DNA by centrifugation using the methods described herein;

FIG. 4 is a schematic diagram of the ultra-sensitive detection methods described herein.

FIG. 5 is a schematic diagram of the ultra-sensitive detection methods described herein.

FIG. 6 is a schematic diagram of the ultra-sensitive detection methods described herein.

FIG. 7 is a schematic diagram of the ultra-sensitive detection methods described herein.

FIG. 8 is a schematic diagram of the ultra-sensitive detection methods described herein.

FIG. 9 is a schematic diagram of the ultra-sensitive detection methods described herein.

FIGS. 10A and 10B show selective lysis of leukocytes leaving Borrelia intact (FIG. 4A) and lysis of Borrelia (FIG. 4B).

FIG. 11 is a graph showing species ID of Borrelia via DIANAs.

FIG. 12 is a graph showing detection of Borrelia directly from blood.

FIG. 13 is a graph showing ultra-sensitive detection and species identification of Borrelia directly from blood.

FIGS. 14A and 14B are graphs showing ultra-sensitive detection of E. faecium directly from blood.

DETAILED DESCRIPTION

Described herein are methods, compositions, and kits for ultrasensitive detection, identification, monitoring, and evaluation of microorganisms, e.g., pathogens such as Borrelia, in a sample from a subject by detecting the genetic material of the microorganisms. These methods, devices, and kits may employ DNA Invading Artificial Nucleic Acids (DIANAs) and novel DIANAs are disclosed herein. Whereas certain known methods in the art rely on hybridization to detect microbial DNA, which has difficulty discriminating among highly similar sequences with high confidence, DIANAs have specificity down to single base-pair resolution, allowing the differentiation of highly homologous sequences.

These methods, devices and kits are particularly useful for ultrasensitive detection of microorganisms. As is used herein, “ultrasensitive detection” is the capability to detect a microbial load at or below 10 cells/ml or 10 CFU/ml of sample. It should be noted that this does not preclude one from being able to detect higher microbial loads as well, however a capability to achieve ultrasensitive detection is a highly sought-after capability where microbial loads evident in clinical samples may, for a meaningful portion of the patient population, require one to detect below 10 cells/ml or 10 CFU/ml, and to do so reliable and consistently. The methods described herein achieve this, in part, through efficient removal (or elimination) of eukaryotic cells, e.g. white blood cells, from large blood volumes prior to processing of microbial DNA in the assays described herein.

Methods in the art generally are not capable of detecting such low levels of microorganisms and commonly use culturing to increase microbial levels. One such family (i.e. genus) of microorganisms is Borrelia, the causative agent of, among other diseases, Lyme disease. The methods presented herein further provide for the ultrasensitive detection of Borrelia from large sample (or specimen) volumes, in part, through (1) specific eukaryotic cell lysis reagents that allow for the selective lysis of eukaryotic cells while leaving microbial cells (e.g. Borrelia), which may be highly sensitive to cell lysis, intact, thereby allowing the removal or depletion of the immense amount of human DNA from the sample, (2) lysis of the microbial cells, (3) isolation and purification of the microbial DNA, (4) enzymatic amplification (e.g. polymerase chain reaction or PCR) of the microbial DNA, and (5) detection, where the use of highly analytically specific DIANAs is advantageous.

The methods, compositions, and kits described herein are particularly useful in the context of evaluating blood samples and evaluating subjects for the presence or progression of Lyme disease, and other infections having low microbial loads. Whole blood is a complex solution that contains multiple cell types such as leukocytes, erythrocytes, and thrombocytes, as well as naturally occurring organic and inorganic components. The blood components can hinder (and may even completely prevent or inhibit) additional or downstream processing of DNA and/or RNA, such as, e.g., enzymatic PCR or isothermal amplification. Additionally, anticoagulants and preservatives, which are commonly used during bodily fluid sample collection, can further interfere with enzymatic or other process. Assaying blood can also require large volumes due to the low frequency (low loads) of microorganisms in Lyme disease as well as in other invasive infections. The methods, compositions, and kits described herein provide for sensitive and accurate evaluation of microorganisms in blood samples. As is described herein, the methods, compositions, and kits are particularly useful for identifying infections with Borrelia.

The methods, kits, and devices described herein may be useful, for example, for clinical purposes (e.g., diagnosing a disease or aliment via the presence of a specific pathogen, e.g., Borrelia), or for research purposes (e.g., for monitoring the changes in the load (i.e. concentration) of one or more pathogens, e.g. Borrelia, within a sample over time due to the addition and/or administration of a compound). Because the approach described herein, among other things, does not require culturing and uses large input volumes, human DNA depletion, anion exchange isolation of microbial genomic material, and DIANAs, it offers significant performance advantages over the art including, for example, improved kinetics, sensitivity, specificity, and dynamic range.

The various aspects and embodiments of the present technology that are introduced above and discussed in greater detail below may be implemented in any number of ways, and as described herein, are not limited to any particular manner of implementation. Examples of specific implementations and applications are provided primarily for illustrative purposes.

As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. For example, reference to “a cell” includes a combination of two or more cells, and the like.

As used herein, “about” will be understood by persons of ordinary skill in the art and will vary to some extent depending upon the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill in the art, given the context in which it is used, “about” will mean up to plus or minus 10% of the particular term.

As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same

DNA Invading Artificial Nucleic Acids (DIANAs)

In some embodiments, DNA Invading Artificial Nucleic Acids (DIANAs) are used to detect microbial genetic materials.

A common method for detection of DNA is to use a complementary strand of DNA to hybridize to single-stranded DNA (ssDNA). An alternative method is to “invade” double-stranded, or duplex, DNA (dsDNA). Invasion requires a nucleic acid which can out-compete the complementary strand that is already present in the dsDNA, e.g., a DIANA.

As is used herein, a “DIANA” refers to any oligonucleotide capable of outcompeting a complementary strand of, e.g. invading, a double stranded DNA molecule to create a stable, hybrid, structure. In some embodiments, a DIANA, has increased affinity to a natural nucleic acid (i.e. DNA) to a level such it can preferentially ‘invade’ a long dsDNA molecule and create, in a highly localized manner, a triplex structure (i.e. DNA₂/DIANA). DIANAs, if employed for hybridization may not outperform other nucleic acids in terms of specificity (and likely will not due to the high levels of affinity), but rather these molecules are unique in that they can identify a target sequence within the long molecule that is maintained in dsDNA form.

As used herein, the term “invasion” refers to the sequence-mediated binding of DIANAs to genomic material (e.g., RNA or DNA) which is in duplex, or double-stranded, form. Similar to that which is common in the field of molecular biology, sequence recognition is through Watson-Crick basepairing rules, while not ruling out alternative mechanisms such as, but not limited to, Hoogstein and reverse-Hoogstein base-pairing rules. Invasion is highly specific as the DNA strand complementary to the DIANA/DNA hybrid remains only a few nanometers away—and competition is fierce. Indeed, in many cases if but a single mismatch is present in this hybrid, the DIANA is kicked out of the duplex DNA, as the hybrid complex is energetically unfavorable. A perfect matching DIANA, in contrast, forms a stable DIANA-DNA structure. This process can be visualized as FIG. 1 . Without wishing to be bound by theory, the physical rationale behind this specificity is as follows. During invasion, a localized ‘bubble’ within the duplex DNA is formed, allowing the DIANA oligonucleotide to bind to a specific sequence along one of the two DNA strands. Throughout, the DNA complement to that sequence remains on the opposing strand, as the DNA is not denatured. Thus, if a single mismatch between the DNA and the DIANA probe is evident, the opposing strand can ‘snap-back’ and ‘kick-out’ the DIANA. It is this consistent and localized energetic battle between the DIANA oligonucleotide and the DNA complement which make the invasion process immensely specific.

Commonly used structures and chemistries for DIANAs are known in the art and disclosed, e.g., in Egholm et al. (Nature, 1993, 365(6446), 566-568), Egholm et al. (Journal of the American Chemical Society, 1992, 114, 1895-1897), Peffer et al. (Proceedings of the National Academy of Sciences of the United States of America, 1993, 90(22), 10648-10652), Nielsen, P. E. (Current opinion in biotechnology, 1999, 10(1), 71-75), Kuhn et al. (Nucleic Acids Research, 1998, 26(2), 582-587), Lohse et al. (Proceedings of the National Academy of Sciences of the United States of America, 1999, 96(21), 11804-11808), Kutyavin et al. (Biochemistry, 1996, 35(34), 11170-11176), Demidov et al. (Proceedings of the National Academy of Sciences of the United States of America, 2002, 99(9), 5953-5958), Dragulescu-Andrasi et al. (Journal of the American Chemical Society, 2006, 128, 10258-10267), Rapireddy et al. (Journal of the American Chemical Society, 2007, 129, 15596-15600), Chenna et al. (ChemBioChem., 2008, 9, 2388-2391), He et al. (Journal of the American Chemical Society, 2009, 131, 12088-12090), Rapireddy et al. (Biochemistry 2011, 50, 3913-3918), WO 2012138955 A2, Eman et al. (Nucleic Acids Research, 2011, 39, 3), Sun et al. (Biochemistry, 2004, 43, 14, 4160-4169), Moreno et al. (Nucleic Acids Research, 2013, 1, 41, 3257-3273), Sau et al. (Organic and Biomolecular Chemistry, 2010, 9).

In some embodiments, the DIANA binds to double stranded DNA or RNA. In some embodiments, the DIANA binds to a predominantly single-stranded DNA or RNA. It is to be understood that the process of DIANA invasion to a DNA or RNA molecule may take place despite the DNA and/or RNA being predominantly single-stranded due to the presence of secondary structures, such as, but not limited, to hairpins. It is to be understood that the process of ‘invasion’ is localized, and the local conditions are those which dictate whether the process is inherently hybridization or invasion based.

A number of methods are known to those of skill in the art to create this increase in specificity and thus create DIANAs such as peptide nucleic acids (PNAs), locked nucleic acids (LNAs), bridged nucleic acids (BNA). Indeed, DIANAs are not limited to a specific chemistry, but rather achieve a physical process by any of a variety of means. The process where identification of a ‘long’ dsDNA molecule is completed via the creation of a localized structure that is different to the rest of the molecule (i.e. triplex).

It is to be understood that no one class of DIANAs (PNAs, LNAs, BNAs) necessarily demonstrate a higher sequence specificity or affinity. The overall enhanced sequence specificity and affinity of DIANAs in relation to DNA hybridization is independent of the class of DIANA used but is a function of the invasion process. While yPNA triplex formation is demonstrated herein, given the state of the art, it is to be understood that other artificial nucleic acids capable of invasion could utilize some or all of the sequences disclosed to achieve the same. DIANAs are, inherently, artificial in nature.

In some embodiments, a DIANA comprises one or more modified nucleotides. In some embodiments, the DIANA is or comprises peptide nucleic acids (PNAs), locked nucleic acids (LNAs), and/or bridged nucleic acids (BNA). In some embodiments, the DIANAs take the form of a specialized type or class of Peptide Nucleic Acids (PNAs), Locked or Bridged Nucleic Acids (LNAs and/or BNAs).

In some embodiments, DIANAs take the form of a specialized type or class of Peptide Nucleic Acids (PNAs). In some embodiments, the DIANAs are not limited to a specific class of PNAs. PNAs, by far are the most studied examples of artificial nucleic acids that may be used as DIANAs. In PNAs, the negatively charged sugar-phosphodiester backbone found in DNA/RNA is replaced by a neutral N-(2-aminoethyl) glycine backbone. Briefly, the negative charges along the backbone of double-stranded DNA/RNA repel one another, overcome by the Watson-Crick pairing and stacking interactions. By replacing the negatively charged backbone found in natural nucleic acids with one that is neutral, PNAs avoids that repulsion and, in theory, can bind with a greater affinity to a ssDNA. This increased affinity (i.e. PNA/DNA hybrid vs dsDNA) manifests itself by having a higher melting temperature of roughly 2-4° C. per PNA monomer. However, as is common in many systems (particularly biological ones), with increased affinity comes decreased analytical specificity (or in the case of PNAs, sequence specificity). Without wishing to be bound by theory, PNAs are notoriously ‘sticky’, and binding conditions need to be optimized to attain a ‘reasonable’ level of sequence specificity.

Within PNAs multiple strategies have been discussed to enable dsDNA invasion including bis-PNA, pc-PNA (with or without 2,6-diaminopurines and 2-thiouracils), yPNA, PNA2-DNA, incorporation of artificial nucleobases such as the use of a 9-(2-guanidinoethoxy) phenoxazine, or the incorporation of a terminally linked acridine moiety. γPNA, is but one specific class among many DIANAs. γPNAs are preferred in that they provide significantly relaxed sequence constraints suitable for invasion in contrast other DIANA classes. γPNA achieve the required affinity to dsDNA as they are, via a chemical modification made to the γ-site along the peptide like backbone, a highly-stable, chiral, structure; one mimicking that of dsDNA—a right-handed helix. By doing this, the energy penalty paid due to the loss of entropy is significantly reduced when transitioning of an unbound γPNA to one that is bound to the dsDNA.

γPNAs are oligonucleotides, comprised of monomers which make up the sequence composition for that oligonucleotide. By way of example by not by way of limitation, the γPNA oligonucleotide with a sequence AGTCAG will be comprised for two ‘A’ monomers, two ‘G’ monomers, a single ‘T’ monomer, and a single ‘C’ monomer. A γPNA oligonucleotide is a specific class of PNA oligonucleotide wherein at least a single monomer contains a chiral stereo-center at the gamma-position of the monomer backbone (herein a ‘gamma-modified monomer’). A PNA oligonucleotide that is pre-oriented structurally into a right-handed helix is energetically favored to perform duplex DNA invasion. In some embodiments, the microbial DNA is detected using γPNA as taught in WO 2013/176992, the contents of which are incorporated by reference in its entirety.

In some embodiments, the oligonucleotide contains more than 5% gamma-modified monomers, more than 10% gamma-modified monomers, more than 25% gamma-modified monomers, more than 50% gamma-modified monomers, more than 75% gamma-modified monomers, or 100% gamma-modified monomers. Suitable modifications at the gamma-site are well known to those skilled in the art and include by way of example, but not by way of limitation, non-polar groups such as methyl groups, ethyl group, etc, or polar groups such as ethylene glycol-based groups, or semi-polar groups, such as those which are ester based.

In some embodiments, the DIANA oligonucleotide may include one or more artificial nucleobases such as, but not limited to pseudo-cytosines, guanidinium G-clamps, diaminopurines, inosines, etc. It is to be understood, that those skilled in the art may utilize artificial or unnatural bases for a number of reasons. Notwithstanding the above, it is the base-pairing rules which dictate if binding (invasion) will occur or not. It is thus to be understood that, in a non-limiting example, the use of a pseudo-cytosines in a DIANA oligonucleotide in place of a cytosine is defined as a homologous sequence.

While one would consider DNA to be a hydrophilic molecule, the entire molecule is not, rather the charged phosphate-sugar backbone induces its overall hydrophilicity while the nucleobases are by themselves are quite hydrophobic. Given that one strategy for the development of DIANA-oligomers calls for the elimination of charge from the backbone to reduce repulsion and increase the its binding affinity, it is well accepted that DIANA-oligomers (and many artificial nucleic acids in general) are rather hydrophobic. Accordingly, in some embodiments, the DIANAs described herein are rather hydrophobic.

In some embodiments, the DIANAs described herein incorporate chemistry to reduce the hydrophobicity of the DIANA molecule. Methods to reduce the hydrophobicity of a DIANA molecule have largely followed the basic peptide-design principles (i.e. incorporate a hydrophilic residue, typically a Lysine, on one or both ends of the oligonucleotide). Thus, in some embodiments, the DIANAs described herein comprise a hydrophilic amino acid at the end of the oligonucleotide. In some embodiments, the DIANAs described herein comprise a hydrophilic amino acid at the 3′ end of the oligonucleotide. In some embodiments, the DIANAs described herein comprise a hydrophilic amino acid at the C-terminus of the oligonucleotide. In some embodiments, the DIANAs described herein comprise a hydrophilic amino acid at the N-terminus of the oligonucleotide.

In some embodiments, the DIANAs described herein comprise a hydrophilic amino acid at the 5′ end and the 3′ end of the oligonucleotide. In some embodiments, the DIANAs described herein comprise a hydrophilic amino acid at the N-terminus and the C-terminus of the oligonucleotide. In some embodiments, a hydrophilic amino acid is selected from Ser, Thr, Cys, Tyr, Asn, Gln, Asp, Glu, Lys, Arg, or His.

WO2012138955, which is incorporated herein by reference in its entirety, discloses a method in which hydrophilic moieties are incorporated along the backbone of the artificial nucleic acid (see paragraph [0091]). In contrast, paragraph of the application is specifically identified as less favorable (while still addressing the hydrophobicity issue) as it reduces sequence specificity. In contrast, our experimental results clearly indicate that at least in the case of “the conjugation of PEG to one of the oligomer termini” provides exceptional results without any detrimental side-effects. Accordingly, in some embodiments, the DIANAs described herein comprise one or more PEG moieties at either the C-terminus or the N-terminus of the oligonucleotide. In some embodiments, the DIANAs described herein comprise one or more PEG moieties at the C-terminus of the oligonucleotide. In some embodiments, the DIANAs described herein comprise one or more PEG moieties at the N-terminus of the oligonucleotide. In some embodiments, the DIANAs described herein comprise one or more PEG moieties at the C-terminus and the N-terminus of the oligonucleotide.

In some embodiments, use of DIANAs is advantageous for long amplicons (e.g., amplicons between about 400 to 4000 bp). It is to be understood, that DIANAs, in some embodiments, could be used in DNA/RNA hybridization processes. However, we identify improved performance when experimental conditions are those which favor invasion in-place of hybridization.

In some embodiments, the DIANA target genetic material from a microorganism. In some embodiments, the DIANA targets genetic material from a bacteria, e.g., a Gram positive or a Gram negative bacteria. In some embodiments, the DIANA targets genetic material from a fungi. In some embodiments, the oligonucleotide sequences for DIANAs useful in Borrelia identification are as shown in Tables 1-33 below. In some embodiments, the sequences for PCR primers useful in the amplification of a specific Borrelia gene, omp66 (or P66) or fla are as shown in Tables 34 and 35 below.

TABLE 1 Seq. Gene/ ID DIANA Sequence Target Plasmid 1 GTTTGATCCTGGCTTAG Broad-Borrelia 16S 2 GCTTAGAACTAACGCTG Broad-Borrelia 16S 3 ACGCTGGCAGTGCGTCT Broad-Borrelia 16S 4 AGCTTCGCTTGTAGATG Broad-Borrelia 16S 5 TAGATGAGTCTGCGTCT Broad-Borrelia 16S 6 TGATAAGTAACCGGCCT Broad-Borrelia 16S 7 CGGAGCGACACTGCGTG Broad-Borrelia 16S 8 TTCTTTTATAAATGAGG Broad-Borrelia 16S 9 ATGAGGAATAAGCTTTG Broad-Borrelia 16S 10 GCTTTGTAGGAAATGAC Broad-Borrelia 16S 11 GATGACGTTAATTTATG Broad-Borrelia 16S 12 TTTATGAATAAGCCCCG Broad-Borrelia 16S 13 GCGAGCGTTGTTCGGGA Broad-Borrelia 16S 14 TCGGGATTATTGGGCGT Broad-Borrelia 16S 15 GATATATAAGTCTATGC Broad-Borrelia 16S 16 CTATGCATAAAATACCA Broad-Borrelia 16S 17 CTATGTTGGAAACTATA Broad-Borrelia 16S 18 ACTATATGTCTAGAGTC Broad-Borrelia 16S 19 GAGGAAGTTAGAATTTC Broad-Borrelia 16S 20 AATTTCTGGTGTAAGGG Broad-Borrelia 16S 21 TAAGGGTGGAATCTGTT Broad-Borrelia 16S 22 GGCGAACTTCTGGGTCA Broad-Borrelia 16S 23 GATGCACACTTGGTGTT Broad-Borrelia 16S 24 GGTGTTAACTAAAAGTT Broad-Borrelia 16S 25 AAAGTTAGTACCGAAGC Broad-Borrelia 16S 26 TTAGAGATAATTATTCC Broad-Borrelia 16S 27 TATTCCCCGTTTGGGGT Broad-Borrelia 16S 28 TGGGGTCTATATACAGG Broad-Borrelia 16S 29 TGTGAGGTGTTGGGTTA Broad-Borrelia 16S 30 CAACCCTTGTTATCTGT Broad-Borrelia 16S 31 ATCTGTTACCAGCATGT Broad-Borrelia 16S 32 ATAAGACTGCCGGTGAT Broad-Borrelia 16S 33 TGGCCTGTACAAAGCGA Broad-Borrelia 16S 34 ATCGTATATCAGAATGA Broad-Borrelia 16S 35 TCCTGGCTTAGAACTAA Broad-Borrelia 16S 36 AACTAACGCTGGCAGTG Broad-Borrelia 16S 37 CGCTTGTAGATGAGTCT Broad-Borrelia 16S 38 GAATAAGGCTTTGTAGG Broad-Borrelia 16S 39 CGTTAATTTATGAATAA Broad-Borrelia 16S 40 CGTTGTTCGGGATTATT Broad-Borrelia 16S 41 TGGTGTAAGGGTGGAAT Broad-Borrelia 16S 42 CACACTTGGTGTTAACT Broad-Borrelia 16S 43 CTTGTTATCTGTTACCA Broad-Borrelia 16S

TABLE 2 Seq. Gene/ ID DIANA Sequence Target Plasmid 44 AATAAGGTCAGTTAATT B. burgdorferi 16S 45 CAACTGTGGACCTATGT B. burgdorferi 16S

TABLE 3 Seq. Gene/ ID DIANA Sequence Target Plasmid 46 ACGGAATGTAGCAATAC B. afzelli 16S 47 CAATACATTTAGTGGCG B. afzelli 16S 48 CTAGAAATAGTAGCTAA B. afzelli 16S

TABLE 4 Seq. Gene/ ID DIANA Sequence Target Plasmid 49 ATTATTCTAACCCGCAA B. mayonii 16S

TABLE 5 Seq. Gene/ ID DIANA Sequence Target Plasmid 50 GTGGATGATCTACCTAC B. garinii 16S 51 ACCTACGAGATGGGGAT B. garinii 16S 52 CAACTGTGGAACTATGT B. garinii 16S

TABLE 6 Seq. Gene/ ID DIANA Sequence Target Plasmid 53 TCATAATACATCAGCTA Broad-Borrelia fla 54 CAGCTATTAATGCTTCA Broad-Borrelia fla 55 TTAATGCTCAAATAAGA Broad-Borrelia fla 56 AATAGAATTGCTGATCA Broad-Borrelia fla 57 AATATAACCAAATGCAC Broad-Borrelia fla 58 TTCTCCTGTTAATGTTA Broad-Borrelia fla 59 GAAAATGCTATTAGAAT Broad-Borrelia fla 60 TAGAATGATAAGTGATC Broad-Borrelia fla 61 GCAAATTTAGGTGCTTT Broad-Borrelia fla 62 TGCTTTCCAAAATAGAC Broad-Borrelia fla 63 ATCTTATGCTCAAATAA Broad-Borrelia fla 64 GCTACAATGACAGATGA Broad-Borrelia fla 65 CTGCAATGGCAATGATT Broad-Borrelia fla 66 GTTTTGTCATTGCTTAG Broad-Borrelia fla 67 TACATCAGCTATTAATG Broad-Borrelia fla 68 TGCTATTAGAATGATA Broad-Borrelia fla 69 TTTAGGTGCTTTCCAAA Broad-Borrelia fla

TABLE 7 Seq. Gene/ ID DIANA Sequence Target Plasmid  70 TAATGGCATTAACGCTG B. burgdorferi fla  71 ACGCTGCTAATCTTAGT B. burgdorferi fla  72 TTTCTAGTGGGTACAGA B. burgdorferi fla  73 ATGATGCTGCTGGCATG B. burgdorferi fla  74 GGCATGGGAGTTTCTGG B. burgdorferi fla  75 CTAGAAATACTTCAAAG B. burgdorferi fla  76 TCAAAGGCTATTAATTT B. burgdorferi fla  77 AAGTCTTAGTAAGAATG B. burgdorferi fla  78 ATGCACATGTTATCAAA B. burgdorferi fla  79 ATCAAACAAATCTGCTT B. burgdorferi fla  80 GGGTCTCAAGCGTCTTG B. burgdorferi fla  81 GTCTTGGACTTTAAGAG B. burgdorferi fla  82 CCAAGATGAAGCTATTG B. burgdorferi fla  83 TCTGGTGAGGGAGCTCA B. burgdorferi fla  84 AGCTCAAACTGCTCAGG B. burgdorferi fla  85 CTCAGGCTGCACCGGTT B. burgdorferi fla  86 CCGGTTCAAGAGGGTGT B. burgdorferi fla  87 ACAGTTGATGCCAATAC B. burgdorferi fla  88 CAATACATCACTTGCTA B. burgdorferi fla  89 ATGCAATTGAAAATCTA B. burgdorferi fla  90 TGGCAGCAACAACTAAT B. burgdorferi fla  91 TTTAACACAATCTGCAA B. burgdorferi fla  92 ATGATTGCGCAGGCTAA B. burgdorferi fla  93 GGCTAATCAAGTTCCCC B. burgdorferi fla  94 CATTAAACGCTGCTAATC B. burgdorferi fla  95 AATACTTCAAAGGCTAT B. burgdorferi fla  96 ATGTTATCAAACAAATC B. burgdorferi fla  97 TCAAGCGTCTTGGACTT B. burgdorferi fla  98 GAGGGAGCTCAAACTGC B. burgdorferi fla  99 AACTGCTCAGGCTGCAC B. burgdorferi fla 100 CTGCACCGGTTCAAGAG B. burgdorferi fla 101 TGATGCCAATACATCAC B. burgdorferi fla 102 GCGCAGGCTAATCAAGT B. burgdorferi fla

TABLE 8 Seq. Gene/ ID DIANA Sequence Target Plasmid 103 GCTTCAAGAAATAATGC B. afzelli fla 104 TAATGCCATTAATGCTG B. afzelli fla 105 CTTAGTAAAACCCAAGA B. afzelli fla 106 CCAAGAGAAGCTTTCTA B. afzelli fla 107 TTTCTAGTGGTTATAGA B. afzelli fla 108 TATAGAATTAATCGAGC B. afzelli fla 109 TTCTGGCAAGATTAATG B. afzelli fla 110 ATAAGAGGCTTATCACA B. afzelli fla 111 GGAACGTATTCAGACTC B. afzelli fla 112 AGACTCAGACAGAGGTT B. afzelli fla 113 GAGGTTCTATACAGATT B. afzelli fla 114 TGATCAGGCTCAATATA B. afzelli fla 115 CATCACTTTCAGGATCT B. afzelli fla 116 TTCTTGGACTTTAAGAG B. afzelli fla 117 TCAAGATGAAGCAATTG B. afzelli fla 118 CAATTGCTGTAAATATT B. afzelli fla 119 CAAATCTTTTTGCTGGT B. afzelli fla 120 GCTGGTGAGGGAGCTCA B. afzelli fla 121 AGCTCAAGCTGCTCAGG B. afzelli fla 122 CTCAGGCTGCACCTGTT B. afzelli fla 123 CCTGTTCAAGAGGGTGC B. afzelli fla 124 CAGCAACCAACACCTGC B. afzelli fla 125 ACCTGCTACAGCACCTA B. afzelli fla 126 CACCTACTCAAGGTGGA B. afzelli fla 127 ATGTTACAACCACAGTT B. afzelli fla 128 TCTATAAAGAATAGCAC B. afzelli fla 129 TAGCACTGAGTATGCTA B. afzelli fla 130 ATGCTATTGAAAATCTA B. afzelli fla 131 TTTAACTCAATCTGCAA B. afzelli fla 132 TTCCTCAATATGTTTTG B. afzelli fla 133 AGAAATAATGCCATTA B. afzelli fla 134 AAAACCCAAGAGAAGC B. afzelli fla 135 CAGAAGCTTTCTAGTGG B. afzelli fla 136 TAGTGGTTATAGAATTA B. afzelli fla 137 GTATTCAGACTCAGACA B. afzelli fla 138 AGACAGAGGTTCTATAC B. afzelli fla 139 ATGAAGCAATTGCTGTA B. afzelli fla 140 CTTTTTGCTGGTGAGGG B. afzelli fla 141 GAGGGAGCTCAAGCTGC B. afzelli fla 142 AGCTGCTCAGGCTGCAC B. afzelli fla 143 GCTGCACCTGTTCAAGA B. afzelli fla 144 CCAACACCTGCTACAGC B. afzelli fla 145 CTACAGCACCTACTCAA B. afzelli fla 146 AAGAATAGCACTGAGT B. afzelli fla 147 CTGAGTATGCTATTGAA B. afzelli fla

TABLE 9 Seq. Gene/ ID DIANA Sequence Target Plasmid 148 ATGCTGCTAATCTTAGC B. mayonii fla 149 CTTAGCAAAACTCAAGA B. mayonii fla 150 TTTCTAGTGGATACAGA B. mayonii fla 151 GGTATGGGAGTTTCTGG B. mayonii fla 152 CTAGAAATACTTCAAAA B. mayonii fla 153 TCAAAAGCCATTAATTT B. mayonii fla 154 TAACACACCATCATCAC B. mayonii fla 155 GGGTCTCAAGCTTCTTG B. mayonii fla 156 TCAAGATGAAGCTATTG B. mayonii fla 157 TCTGGTGAGGGAACTCA B. mayonii fla 158 AACTCAAACTGCTCAGG B. mayonii fla 159 CTCAGGTTGCGCCTGTT B. mayonii fla 160 AGATGAGGTTGTAGCTG B. mayonii fla 161 TAGCTGCAACAACTAAT B. mayonii fla 162 ACTAATAGTATCTTAAC B. mayonii fla 163 CTTAACACAATCTGCAA B. mayonii fla 164 TTCCTCAGTATGTTTTG B. mayonii fla 165 CTAATCTTAGCAAAAC B. mayonii fla 166 ATACTTCAAAAGCCATT B. mayonii fla 167 GAGGGAACTCAAACTGC B. mayonii fla 168 AACTGCTCAGGTTGCGC B. mayonii fla 169 AGGTTGTAGCTGCAACA B. mayonii fla 170 CAACAACTAATAGTATC B. mayonii fla 171 AGTATCTTAACACAATC B. mayonii fla

TABLE 10 Seq. Gene/ ID DIANA Sequence Target Plasmid 172 TCAAGAGAAGCTTTCTA B. garinii fla 173 TTTCTAGTGGTTACAGA B. garinii fla 174 TACAGAATTAATAGAGC B. garinii fla 175 TAGAGCTTCTGATGATG B. garinii fla 176 TTCTGGGAAGATTAATG B. garinii fla 177 ATAAGAGGTTTATCACA B. garinii fla 178 AATCAGGTAACGGTACA B. garinii fla 179 GGTACATATTCAGACGC B. garinii fla 180 AGAGCAACTTACAGATG B. garinii fla 181 TTGGAATGCAACCTGCG B. garinii fla 182 CCTGCGAAAATCAACAC B. garinii fla 183 CAACACACCAGCGTCAC B. garinii fla 184 CGTCACTTTCAGGATCT B. garinii fla 185 TCAAGATGAAGCGATTG B. garinii fla 186 CGATTGCTGTAAATATT B. garinii fla 187 AATATTTATGCTGCTAA B. garinii fla 188 TGCTAATGTTGCAAATC B. garinii fla 189 CAAATCTATTCTCTGGC B. garinii fla 190 TCTGGCGAAGGAGCTCA B. garinii fla 191 AGCTCAGGCTGCTCAGA B. garinii fla 192 CTCAGACTGCACCTGTT B. garinii fla 193 ACCTGCTACAGCGCCTT B. garinii fla 194 CGCCTTCTCAGGGTGGA B. garinii fla 195 ACAGTTGACGCTAATAC B. garinii fla 196 TAATACATCTCTTGCTA B. garinii fla 197 ATAGACTTGAGTCTATA B. garinii fla 198 TCTATAAAGGATAGTAC B. garinii fla 199 TAGTACTGAGTATGCTA B. garinii fla 200 ATGCTATTGAAAACCTA B. garinii fla 201 AACCTAAAAGCATCTTA B. garinii fla 202 ACTAATAGTATTTTGAC B. garinii fla 203 TTTGACACAATCTGCAA B. garinii fla 204 ATGATTGCGCAAGCTAA B. garinii fla 205 AGCTAATCAAGTTCCCC B. garinii fla 206 GAAGCTTTCTAGTGGTT B. garinii fla 207 GTGGTTACAGAATTAAT B. garinii fla 208 AATTAATAGAGCTTCTG B. garinii fla 209 GGTAACGGTACATATTC B. garinii fla 210 TATTCAGAGCAACTTAC B. garinii fla 211 TGCAACCTGCGAAAATC B. garinii fla 212 CACCAGCGTCACTTTCA B. garinii fla 213 TGAAGCGATTGCTGTAA B. garinii fla 214 CTGTAAATATTTATGCT B. garinii fla 215 TATGCTGCTAATGTTGC B. garinii fla 216 TGTTGCAAATCTATTCT B. garinii fla 217 CTATTCTGGCGAAGGA B. garinii fla 218 GAAGGAGCTCAGGCTGC B. garinii fla 219 GGCTGCTCAGACTGCAC B. garinii fla 220 CTGCACCTGCTACAGC B. garinii fla 221 CTACAGCGCCTTCTCAG B. garinii fla 222 GACGCTAATACATCTCT B. garinii fla 223 CTTGAGTCTATAAAGGA B. garinii fla 224 AAGGATAGTACTGAGTA B. garinii fla 225 CTGAGTATGCTATTGAA B. garinii fla 226 TTGAAAACCTAAAAGCA B. garinii fla 227 AGTATTTTGACACAATC B. garinii fla 228 GCGCAAGCTAATCAAGT B. garinii fla

TABLE 11 Seq. Gene/ ID DIANA Sequence Target Plasmid 229 TTTATTGGGAATAGGTC Broad-Borrelia OspA 230 TAGGTCTAATATTAGCC Broad-Borrelia OspA 231 TTAGCCTTAATAGCATG Broad-Borrelia OspA 232 AAAATGTTAGCAGCCTT Broad-Borrelia OspA 233 TGGGAATAGGTCTAATA Broad-Borrelia OspA 234 CTAATATTAGCCTTAAT Broad-Borrelia OspA

TABLE 12 Seq. Gene/ ID DIANA Sequence Target Plasmid 235 GAAAAACAGCGTTTCAG B. burgdorferi OspA 236 TTTCAGTAGATTTGCCT B. burgdorferi OspA 237 TTGCCTGGTGAAATGAA B. burgdorferi OspA 238 AGACGGCAAGTACGATC B. burgdorferi OspA 239 ACGATCTAATTGCAACA B. burgdorferi OspA 240 TCTGATAAAAACAATGG B. burgdorferi OspA 241 CAATGGATCTGGAGTAC B. burgdorferi OspA 242 ACAATTTCTGACGATCT B. burgdorferi OspA 243 CGATCTAGGTCAAACCA B. burgdorferi OspA 244 AAACACTAGTATCAAAA B. burgdorferi OspA 245 TCAAAAAAAGTAACTTC B. burgdorferi OspA 246 AACTTCCAAAGACAAGT B. burgdorferi OspA 247 CAGACGGAACCAGACTT B. burgdorferi OspA 248 AGACTTGAATACACAGG B. burgdorferi OspA 249 TAAAAGGCTATGTTCTT B. burgdorferi OspA 250 GTTCTTGAAGGAACTCT B. burgdorferi OspA 251 AACTCTAACTGCTGAAA B. burgdorferi OspA 252 CTGAAAAAACAACATTG B. burgdorferi OspA 253 CTGTTACTTTAAGCAAA B. burgdorferi OspA 254 TTCAAAATCTGGGGAAG B. burgdorferi OspA 255 GGGAAGTTTCAGTTGAA B. burgdorferi OspA 256 TGACACTGACAGTAGTG B. burgdorferi OspA 257 GTAGTGCTGCTACTAAA B. burgdorferi OspA 258 ACTAAAAAAACTGCAGC B. burgdorferi OspA 259 TGCAGCTTGGAATTCAG B. burgdorferi OspA 260 ATTCAGGCACTTCAACT B. burgdorferi OspA 261 AATTACTGTAAACAGTA B. burgdorferi OspA 262 ATTACAGTACAACAATA B. burgdorferi OspA 263 ACAATACGACTCAAATG B. burgdorferi OspA 264 CAAATGGCACCAAATTA B. burgdorferi OspA 265 GTCAGCAGTTGAAATTA B. burgdorferi OspA 266 CAGCGTTTCAGTAGATT B. burgdorferi OspA 267 GTAGATTTGCCTGGTGA B. burgdorferi OspA 268 CAAGTACGATCTAATTG B. burgdorferi OspA 269 AAAAACAATGGATCTGG B. burgdorferi OspA 270 TTCTGACGATCTAGGTC B. burgdorferi OspA 271 AGGTCAAACACTAGTAT B. burgdorferi OspA 272 AAAGTAACTTCCAAAGA B. burgdorferi OspA 273 GAACCAGACTTGAATAC B. burgdorferi OspA 274 GGCTATGTTCTTGAAGG B. burgdorferi OspA 275 GAAGGAACTCTAACTGC B. burgdorferi OspA 276 TAACTGCTGAAAAAACA B. burgdorferi OspA 277 CTGACAGTAGTGCTGCT B. burgdorferi OspA 278 GCTGCTACTAAAAAAAC B. burgdorferi OspA 279 AAAACTGCAGCTTGGAA B. burgdorferi OspA 280 CTTGGAATTCAGGCACT B. burgdorferi OspA 281 GTACAACAATACGACTC B. burgdorferi OspA

TABLE 13 Seq. Gene/ ID DIANA Sequence Target Plasmid 282 AAAAAACAGCGCTTCAG B. afzelli OspA 283 CTTCAGTAGATTTGCCT B. afzelli OspA 284 TTGCCTGGTGAGATGAA B. afzelli OspA 285 GATGAAAGTTCTTGTAA B. afzelli OspA 286 AGACGGTAAGTACAGTC B. afzelli OspA 287 ACAGTCTAAAGGCAACA B. afzelli OspA 288 TCTGATAAAGACAATGG B. afzelli OspA 289 CAATGGTTCTGGGGTGC B. afzelli OspA 290 GGGTGCTTGAAGGTACA B. afzelli OspA 291 ACAATTGCTGACGATCT B. afzelli OspA 292 CGATCTAAGTAAAACCA B. afzelli OspA 293 AAACCACATTCGAACTT B. afzelli OspA 294 GAACTTTTCAAAGAAGA B. afzelli OspA 295 TCAAGAAAAGTAAGTTC B. afzelli OspA 296 ACAAAACATCAACAGAT B. afzelli OspA 297 ACAGATGAAATGTTCAA B. afzelli OspA 298 AAGGTGAATTGTCTGCA B. afzelli OspA 299 TCTGCAAAAACCATGAC B. afzelli OspA 300 AAAATGGAACCAAACTT B. afzelli OspA 301 AAACTTGAATATACAGA B. afzelli OspA 302 ACTCTTGAAGGAAAAGT B. afzelli OspA 303 ATGATAAAGTAACATTG B. afzelli OspA 304 CCGTTACTTTAAGTAAG B. afzelli OspA 305 GAGAAGTAACAGTTGCT B. afzelli OspA 306 GTTGCTCTTAATGACAC B. afzelli OspA 307 TGACACTAACACTACTC B. afzelli OspA 308 CTACTCAGGCTACTAAA B. afzelli OspA 309 ACTAAAAAAACTGGCGC B. afzelli OspA 310 TGGCGCATGGGATTCAA B. afzelli OspA 311 ATTCAAAAACTTCTACT B. afzelli OspA 312 TCTACTTTAACAATTAG B. afzelli OspA 313 AATTAGTGTTAACAGCA B. afzelli OspA 314 ACTACACAACTTGTGTT B. afzelli OspA 315 TGTGTTTACTAAACAAG B. afzelli OspA 316 AACAAGACACAATAACT B. afzelli OspA 317 AAAATACGACTCCGCAG B. afzelli OspA 318 CCGCAGGTACCAATTTA B. afzelli OspA 319 AATTTAGAAGGCACAGC B. afzelli OspA 320 CACAGCAGTCGAAATTA B. afzelli OspA 321 TAAAAACGCTTTGAAAT B. afzelli OspA 322 CAGCGCTTCAGTAGATT B. afzelli OspA 323 TAGATTTGCCTGGTGAG B. afzelli OspA 324 GGTGAGATGAAAGTTCT B. afzelli OspA 325 TAAGTACAGTCTAAAGG B. afzelli OspA 326 AAAGACAATGGTTCTGG B. afzelli OspA 327 TTCTGGGGTGCTTGAAG B. afzelli OspA 328 TGCTGACGATCTAAGTA B. afzelli OspA 329 AAGTAAAACCACATTCG B. afzelli OspA 330 ACATTCGAACTTTTCAA B. afzelli OspA 331 CATCAACAGATGAAATG B. afzelli OspA 332 AATTGTCTGCAAAAACC B. afzelli OspA 333 GGAACCAAAATGGAACC B. afzelli OspA 334 GTAACAGTTGCTCTTAA B. afzelli OspA 335 CTTAATGACACTAACAC B. afzelli OspA 336 CTAACACTACTCAGGCT B. afzelli OspA 337 CAGGCTACTAAAAAAAC B. afzelli OspA 338 AAAACTGGCGCATGGGA B. afzelli OspA 339 CATGGGATTCAAAAACT B. afzelli OspA 340 AAACTTCTACTTTAACA B. afzelli OspA 341 TTTAACAATTAGTGTTA B. afzelli OspA 342 CAACTTGTGTTTACTAA B. afzelli OspA 343 TTACTAAACAAGACACA B. afzelli OspA 344 CGACTCCGCAGGTACCA B. afzelli OspA 345 GTACCAATTTAGAAGGC B. afzelli OspA 346 GAAGGCACAGCAGTCGA B. afzelli OspA

TABLE 14 Seq. Gene/ ID DIANA Sequence Target Plasmid 347 GAAAAACAGTGTTTCAG B. mayonii OspA 348 TTACCTGGTGAAATTAA B. mayonii OspA 349 AGACGGCAAGTACAGCC B. mayonii OspA 350 TCTGATAAAAATAATGG B. mayonii OspA 351 TAATGGATCTGGAGTAC B. mayonii OspA 352 ACAGTTTCTGACGATCT B. mayonii OspA 353 AAACATTAGTATCAAGA B. mayonii OspA 354 TCAAGAAAAGTAACTTC B. mayonii OspA 355 AACTTCTAAAGACAAGT B. mayonii OspA 356 CTGACGAAACCAGACTT B. mayonii OspA 357 TAAAAGGCTATGCTCTT B. mayonii OspA 358 GCTCTTGAAGGAACTTT B. mayonii OspA 359 AACTTTAACTGCCGAAA B. mayonii OspA 360 CCGAAAAAACAACATTG B. mayonii OspA 361 CTGTTACTTTAAGTAAG B. mayonii OspA 362 AGTAAGAACATTTCAAA B. mayonii OspA 363 TTCAAAATCTGGAGAAG B. mayonii OspA 364 GCTGAGCTTAATGACAC B. mayonii OspA 365 TGACACTGACAGTGCTG B. mayonii OspA 366 GTGCTGCTGCTACTAAA B. mayonii OspA 367 TGGAGCTTGGAATTCAG B. mayonii OspA 368 ATTCAGGCACCTCAACT B. mayonii OspA 369 AATTACTGCTAACAGCA B. mayonii OspA 370 AAAATACGACACAGCTG B. mayonii OspA 371 CAGCTGGCATTAAATTG B. mayonii OspA 372 ATCAGCAGTTGAAATTA B. mayonii OspA 373 AAAAATAATGGATCTGG B. mayonii OspA 374 TTAGTATCAAGAAAAGT B. mayonii OspA 375 AAAGTAACTTCTAAAGA B. mayonii OspA 376 GGCTATGCTCTTGAAGG B. mayonii OspA 377 GAAGGAACTTTAACTGC B. mayonii OspA 378 TAACTGCCGAAAAAACA B. mayonii OspA 379 CTTTAAGTAAGAACATT B. mayonii OspA 380 AACATTTCAAAATCTGG B. mayonii OspA 381 CTTAATGACACTGACAG B. mayonii OspA 382 CTGACAGTGCTGCTGCT B. mayonii OspA 383 CTTGGAATTCAGGCACC B. mayonii OspA 384 ACGACACAGCTGGCATT B. mayonii OspA

TABLE 15 Seq. Gene/ ID DIANA Sequence Target Plasmid 385 GAAAAATAGCGTTTCAG B. garinii OspA 386 TTACCTGGTGAAATGAA B. garinii OspA 387 AGATGGTAAATACAGCC B. garinii OspA 388 GCAACAGTAGACAAACT B. garinii OspA 389 CAAACTTGAGCTAAAAG B. garinii OspA 390 TCTGATAAAAGCAATGG B. garinii OspA 391 CAATGGTTCTGGGGTAC B. garinii OspA 392 GGGTACTTGAAGGTGTA B. garinii OspA 393 AAGCAAAATTAACCATT B. garinii OspA 394 ACCATTTCTGACGATCT B. garinii OspA 395 AAACCACATTTGAAGTT B. garinii OspA 396 TCAAGAAAAGTAAATTC B. garinii OspA 397 AAATTCTAAAGACAAGT B. garinii OspA 398 ATTTAATGCAAAAGGTG B. garinii OspA 399 CAAACGGAAACAGACTT B. garinii OspA 400 TAAAGGGCTTTACTCTT B. garinii OspA 401 ACTCTTGAAGGAACTCT B. garinii OspA 402 AACTCTAACTGCTGACA B. garinii OspA 403 CTGACAAAACAACATTA B. garinii OspA 404 ACATTAACAGTTAAAGA B. garinii OspA 405 TAAAGAGGGCACTGTTA B. garinii OspA 406 CTGTTACTTTAAGCAAG B. garinii OspA 407 TGACACTGACTCTAGCG B. garinii OspA 408 CTAGCGGTACTAAAAAA B. garinii OspA 409 ACAATGGAATTCAAGTA B. garinii OspA 410 CAAGTACTTCTACTTTA B. garinii OspA 411 ACTTTAACAATTAGTGC B. garinii OspA 412 TAGTGCTAACAACAAAA B. garinii OspA 413 AAAGATCTTGTATTTAC B. garinii OspA 414 ATTTACAAAACAAGACA B. garinii OspA 415 ATACGACTCAGCAGCAG B. garinii OspA 416 CAGCAGGAACCACGCTT B. garinii OspA 417 ACGCTTGAAGGCTCCGC B. garinii OspA 418 CTCCGCAGTTGAAATTA B. garinii OspA 419 AAATTAAAACACTTGAC B. garinii OspA 420 CTTGACGAACTTAAAAA B. garinii OspA 421 AGTAGACAAACTTGAGC B. garinii OspA 422 AAAAGCAATGGTTCTGG B. garinii OspA 423 AATTAACCATTTCTGAC B. garinii OspA 424 AAAGTAAATTCTAAAGA B. garinii OspA 425 GGCTTTACTCTTGAAGG B. garinii OspA 426 GAAGGAACTCTAACTGC B. garinii OspA 427 TAACTGCTGACAAAACA B. garinii OspA 428 AAACAACATTAACAGTT B. garinii OspA 429 ACAGTTAAAGAGGGCAC B. garinii OspA 430 GGGCACTGTTACTTTAA B. garinii OspA 431 CTGACTCTAGCGGTACT B. garinii OspA 432 GGAATTCAAGTACTTCT B. garinii OspA 433 CTTCTACTTTAACAATT B. garinii OspA 434 ACAATTAGTGCTAACAA B. garinii OspA 435 TCTTGTATTTACAAAAC B. garinii OspA 436 CTCAGCAGCAGGAACCA B. garinii OspA 437 GAACCACGCTTGAAGGC B. garinii OspA 438 TGAAGGCTCCGCAGTTG B. garinii OspA 439 AGTTGAAATTAAAACAC B. garinii OspA 440 AAACACTTGACGAACTT B. garinii OspA

TABLE 16 Seq. Gene/ ID DIANA Sequence Target Plasmid 441 GAGTCAATTGGTTCTCA B. burgdorferi OspB 442 AACCTTGAAGACTCTAG B. burgdorferi OspB 443 CTCTAGTAAAAAATCAC B. burgdorferi OspB 444 GAAGACTCAGTGTCTTT B. burgdorferi OspB 445 AACAGAGGAAACTCTCA B. burgdorferi OspB 446 GATGCTGACAATGCTAC B. burgdorferi OspB 447 AAGGAAGTCTTGTAGGC B. burgdorferi OspB 448 TGGTGTTCTTAACAGAT B. burgdorferi OspB 449 TGGAACCAGCCTAGAAG B. burgdorferi OspB 450 TGAAGACTCTAGTAAAA B. burgdorferi OspB

TABLE 17 Seq. Gene/ ID DIANA Sequence Target Plasmid 451 TTAGCTTTAATAGCGTG B. afzelli OspB 452 AGCGTGTTCTCAAAAAG B. afzelli OspB 453 GGTTTCTGACAAGAATA B. afzelli OspB 454 AACGAAACTACTAACAC B. afzelli OspB 455 AACTAAAGATCTTGTGT B. afzelli OspB 456 TTGTGTTCTTAACAGAT B. afzelli OspB 457 TTGAGGGCAACCCAAGT B. afzelli OspB 458 TTTAATAGCGTGTTCTCA B. afzelli OspB 459 AAGATCTTGTGTTCTTAA B. afzelli OspB

TABLE 18 Seq. Gene/ ID DIANA Sequence Target Plasmid 460 TTTACTAGGCTTTACTT B. mayonii OspB 461 GAAGACTTAGTGTCTTT B. mayonii OspB 462 ATAGCTCCGGTAAATAT B. mayonii OspB 463 GTTAATGGTTTCTGACG B. mayonii OspB 464 ATGACGCTAGCAACCAA B. mayonii OspB 465 AACAGAGGAAACCCTCA B. mayonii OspB 466 TAAATGACACTGCATCT B. mayonii OspB 467 TGGTGTTCTTAACAGAC B. mayonii OspB 468 CAGTACAAAACTATGAC B. mayonii OspB 469 TGGCACTTCCCTTGAAG B. mayonii OspB

TABLE 19 Seq. Gene/ ID DIANA Sequence Target Plasmid 470 TGTTAAAGGGCCTAATC B. burgdorferi OspC 471 ATTCTAATGCGGTTTTA B. burgdorferi OspC 472 CTGTCATCTATAGATGA B. burgdorferi OspC 473 GATCATTGTTAGCGGGA B. burgdorferi OspC 474 ATCAGTAGAGGTCTTGT B. burgdorferi OspC 475 GAGCTTACAAGCCCTGT B. burgdorferi OspC

TABLE 20 Seq. Gene/ ID DIANA Sequence Target Plasmid 476 GGATTCTGCATCTACTA B. afzelli OspC 477 TTTGTACTGGCTGTTAA B. afzelli OspC 478 TGGATTGAAAGGTCTAG B. afzelli OspC 479 ATCACTAACCAATTCAG B. afzelli OspC 480 GAGCTTACAAACCCTGT B. afzelli OspC

Seq. Gene/ ID DIANA Sequence Target Plasmid 481 CTAAGGAATGTTCCGAA B. mayonii OspC 482 TAGAAACCAATCACACA B. mayonii OspC 483 TGGTAAACATGATGCTA B. mayonii OspC 484 GCTTGTCAACAGAAGCT B. mayonii OspC 485 AATGCTAACTAATTCAG B. mayonii OspC 486 CAGCTTACAAGTCCTGT B. mayonii OspC

TABLE 22 Seq. Gene/ ID DIANA Sequence Target Plasmid 487 TGCAAAAGGACCTAATC B. garinii OspC 488 TTGAGGCTTTGATCTCA B. garinii OspC 489 AATGCTAATGCGGGTCA B. garinii OspC 490 TAAAAGGTTCTCATGCA B. garinii OspC 491 AGCATTAGCTAATTCAG B. garinii OspC 492 GAACTTACAAATCCTGT B. garinii OspC

TABLE 23 Seq. Gene/ ID DIANA Sequence Target Plasmid 493 AAGACGATCCATTCTCA Broad-Borrelia Omp66 494 TTCTCAGCTTACATTAA Broad-Borrelia Omp66 495 ATGACAGATTTTGACTT Broad-Borrelia Omp66 496 AATTCTTGCAAGAGGTA Broad-Borrelia Omp66 497 CCAATAAAAAATCTACT Broad-Borrelia Omp66 498 TTTATCAAATTCTGCAA Broad-Borrelia Omp66 499 CTGCAATTTTAGCATCT Broad-Borrelia Omp66 500 GAATAGATCCTTTTGCA Broad-Borrelia Omp66 501 TTTGCAAGCGATTTTTC Broad-Borrelia Omp66 502 TTTTTCTGTATTTGGAC Broad-Borrelia Omp66 503 TTTCAAAGCTTAAATGT Broad-Borrelia Omp66 504 TTATATCTTGATTATGC Broad-Borrelia Omp66 505 TTAAGACAAAAATCTGT Broad-Borrelia Omp66 506 ATCTGTAGAAAACTATC Broad-Borrelia Omp66 507 ATTATGCAATTCCAATA Broad-Borrelia Omp66 508 GTGCTTTCTTGCAATTC Broad-Borrelia Omp66 509 CAATTCAAAATAGCCTA Broad-Borrelia Omp66 510 AGCCTACAGCGGAAGCT Broad-Borrelia Omp66 511 GATCCATTCTCAGCTTAC Broad-Borrelia Omp66 512 GCAAGCGATTTTTCT Broad-Borrelia Omp66 513 GATCCTTTTGCAAGCGAT Broad-Borrelia Omp66 514 AAGCGATTTTTCTGTATT Broad-Borrelia Omp66 515 CAAAATAGCCTACAGCG Broad-Borrelia Omp66 516 GGATGGATAACATCTAT Broad-Borrelia Omp66 517 AGCTTAAATGTTGAAAT Broad-Borrelia Omp66 518 TGGATAACATCTATCGG Broad-Borrelia Omp66 519 GACAAAAATCTGTAGAA Broad-Borrelia Omp66 520 CAAATTCTGCAATTTTAG Broad-Borrelia Omp66 521 ATCCAAGACCAGGAATA Broad-Borrelia Omp66 522 TGCATTTGATAAAGTTG Broad-Borrelia Omp66 523 GATTAAATGTTGAGTTT Broad-Borrelia Omp66 524 TTATCTTCATAAGTTGA Broad-Borrelia Omp66 525 TATCTTCATAAGTTGAA Broad-Borrelia Omp66 526 ATCTTCATAAGTTGAAA Broad-Borrelia Omp66 527 GCTATCCATCCAAGACC Broad-Borrelia Omp66 528 CTATCCATCCAAGACCA Broad-Borrelia Omp66 529 TATCCATCCAAGACCAG Broad-Borrelia Omp66 530 ATCCATCCAAGACCAGG Broad-Borrelia Omp66 531 TCCATCCAAGACCAGGA Broad-Borrelia Omp66 532 CCATCCAAGACCAGGAA Broad-Borrelia Omp66 533 CATCCAAGACCAGGAAT Broad-Borrelia Omp66 534 ATCCAAGACCAGGAATA Broad-Borrelia Omp66 535 TCCAAGACCAGGAATAA Broad-Borrelia Omp66 536 AATGCAAAATTAGTGGT Broad-Borrelia Omp66 537 ATGCAAAATTAGTGGTT Broad-Borrelia Omp66 538 ATTTTTGTAAGACCAA Broad-Borrelia Omp66 539 GATGCATTTGATAAAGT Broad-Borrelia Omp66 540 ATGCATTTGATAAAGTT Broad-Borrelia Omp66 541 TGCATTTGATAAAGTTG Broad-Borrelia Omp66 542 GCATTTGATAAAGTTGG Broad-Borrelia Omp66 543 TGATGAATGATTAAATG Broad-Borrelia Omp66 544 GATGAATGATTAAATGT Broad-Borrelia Omp66 545 TGAATGATTAAATGTTG Broad-Borrelia Omp66 546 GAATGATTAAATGTTGA Broad-Borrelia Omp66 547 AATGATTAAATGTTGAG Broad-Borrelia Omp66 548 ATGATTAAATGTTGAGT Broad-Borrelia Omp66 549 TGATTAAATGTTGAGTT Broad-Borrelia Omp66 550 GATTAAATGTTGAGTTT Broad-Borrelia Omp66 551 ATTAAATGTTGAGTTTC Broad-Borrelia Omp66 552 TTAAATGTTGAGTTTCC Broad-Borrelia Omp66 553 TAAATGTTGAGTTTCCG Broad-Borrelia Omp66 554 AAATGTTGAGTTTCCGA Broad-Borrelia Omp66 555 AATGTTGAGTTTCCGAT Broad-Borrelia Omp66 556 ATGTTGAGTTTCCGATT Broad-Borrelia Omp66 557 TTGCTCCAATTGCAA Broad-Borrelia Omp66 558 TTAATAGGTCATAAATC Broad-Borrelia Omp66 559 TAAATCCCCATTGAAGC Broad-Borrelia Omp66 560 TGAAGCTATCCATCCAA Broad-Borrelia Omp66 561 GGAATAAGACCTTTCTT Broad-Borrelia Omp66 562 CTTCATAAGTTGAAAGC Broad-Borrelia Omp66 563 GAAAGCTCTGCATTGAG Broad-Borrelia Omp66 564 ATTGAGAGTTTTAAATG Broad-Borrelia Omp66 565 TAAATGATTTTTCAGAG Broad-Borrelia Omp66 566 TCAGAGATTCTCTTTAG Broad-Borrelia Omp66 567 CTTTAGTAGTGGTATGT Broad-Borrelia Omp66 568 GTATGTTGTAAGATTGA Broad-Borrelia Omp66 569 ATATAAGTTTTGTCCAT Broad-Borrelia Omp66 570 GTCCATAGCTAATTCCA Broad-Borrelia Omp66 571 ATTCCAATTCCAACTCC Broad-Borrelia Omp66 572 AACTCCAGATTTTTTAT Broad-Borrelia Omp66 573 TTTTATCTTGTTCACCA Broad-Borrelia Omp66 574 AAGAATCACTTCCTCTA Broad-Borrelia Omp66 575 CCTCTAATTGCCCATGA Broad-Borrelia Omp66 576 CCATGATTCTTTTTCTC Broad-Borrelia Omp66 577 TTTCTCCTTCATCTTTA Broad-Borrelia Omp66 578 TCTTTATTCCAAGCGAA Broad-Borrelia Omp66 579 AGCGAAACCAATACCTA Broad-Borrelia Omp66 580 TACCTATTCCTGCAGAA Broad-Borrelia Omp66 581 ATTATTCGATTTTGGAT Broad-Borrelia Omp66 582 TTTATCTGTGTTTGCTT Broad-Borrelia Omp66 583 TTGCTTTTTTAGATATG Broad-Borrelia Omp66 584 GATATGTGTCCAAATAT Broad-Borrelia Omp66 585 AATCGCTAGCAAATGGA Broad-Borrelia Omp66 586 AATGGATCTATTCCAAG Broad-Borrelia Omp66 587 TCCAAGGTCATAACCTG Broad-Borrelia Omp66 588 AACCTGTTTTTAGTATT Broad-Borrelia Omp66 589 AGTATTAGGTAAGTATT Broad-Borrelia Omp66 590 GTAAGACCAATCTTGTA Broad-Borrelia Omp66 591 ATCCAATGGAGGCTATT Broad-Borrelia Omp66 592 GCTATTATGGATGCATT Broad-Borrelia Omp66 593 AAGTTGGACTTACAAGA Broad-Borrelia Omp66 594 ACAAGATCTGAGTTAGT Broad-Borrelia Omp66 595 GTTAGTAATAGCTGTAT Broad-Borrelia Omp66 596 CTGTATTTTGTACAGTA Broad-Borrelia Omp66 597 ACAGTATATGTTGATGA Broad-Borrelia Omp66 598 TGATGATGAATGATTAA Broad-Borrelia Omp66 599 GAGTTTCCGATTCCCCC Broad-Borrelia Omp66 600 TCCCCCTGAGAGTCCAA Broad-Borrelia Omp66 601 GTCCAAAATTTAATTCA Broad-Borrelia Omp66 602 AATTCAAAAGGGGTTTC Broad-Borrelia Omp66 603 GGTTTCTACAATAACAT Broad-Borrelia Omp66 604 TAACATTTGGATTGTTT Broad-Borrelia Omp66 605 TTGTTTTGTTCTAATAC Broad-Borrelia Omp66 606 GTATTGGTTTCCATTTT Broad-Borrelia Omp66 607 CATTTTAATTGAGTTCC Broad-Borrelia Omp66 608 AGTTCCGTAAATTATGC Broad-Borrelia Omp66 609 TTATGCCTTTATAAGTC Broad-Borrelia Omp66 610 TAAGTCTCATTGTAAGG Broad-Borrelia Omp66 611 GTAAGGGGTTTCTATTT Broad-Borrelia Omp66 612 TTTTTTTCCTCGTCAGA Broad-Borrelia Omp66 613 GTCAGAATCGTCATTGT Broad-Borrelia Omp66 614 CATTGTTTTTTTGATGA Broad-Borrelia Omp66 615 TGATGATTTCTATTACC Broad-Borrelia Omp66 616 ATTACCTGTTCCTATTG Broad-Borrelia Omp66 617 CTATTGCTCCAATTGCA Broad-Borrelia Omp66 618 ATTGCAATCAAAAACTC Broad-Borrelia Omp66 619 AAACTCTATTTGTGGTG Broad-Borrelia Omp66 620 GTGGTGGCAGATTGTAT Broad-Borrelia Omp66 621 TTGTATCCAATTTGAAG Broad-Borrelia Omp66 622 TTGAAGTGTACCTGTTC Broad-Borrelia Omp66 623 CTGTTCTTTTAATTTTG Broad-Borrelia Omp66 624 ATTTTGCTTTTTGAGAA Broad-Borrelia Omp66 625 TGAGAAAATTTTTTGAC Broad-Borrelia Omp66 626 TTTGACCTATATCTCTG Broad-Borrelia Omp66 627 TCTCTGTTCTTACTTGG Broad-Borrelia Omp66 628 ACTTGGAAAACCGTAGT Broad-Borrelia Omp66 629 CGTAGTATTTACTTTGA Broad-Borrelia Omp66 630 CTTTGAATGCCAGTCAT Broad-Borrelia Omp66 631 AGTCATTGGCGCGAAAC Broad-Borrelia Omp66 632 CGAAACTGAATAATGAT Broad-Borrelia Omp66 633 AATGATTCTTGGTTAAA Broad-Borrelia Omp66 634 GTTAAAATCAAAGTTGG Broad-Borrelia Omp66 635 AGTTGGTCATCGACTCC Broad-Borrelia Omp66 636 GACTCCATTTTCAGGTG Broad-Borrelia Omp66 637 CAGGTGGAAATCATATA Broad-Borrelia Omp66 638 ATATTTGCTATGATTTC Broad-Borrelia Omp66 639 GATTTCCCCTAAATCAA Broad-Borrelia Omp66 640 AATATGGCATCTTTTGT Broad-Borrelia Omp66 641 TTTTGTTCCTTGAGCTT Broad-Borrelia Omp66 642 GAGCTTTGAAGCCCACA Broad-Borrelia Omp66 643 CCCACATTTTCTATTTT Broad-Borrelia Omp66 644 TATTTTGATGTAAGCTG Broad-Borrelia Omp66 645 GGGTCGTCTTTTCCTAT Broad-Borrelia Omp66 646 TATTTTTTTCAGGTGCT Broad-Borrelia Omp66 647 GGTGCTTGAAATTTGAT Broad-Borrelia Omp66 648 TTTGATTCCTATCTGGC Broad-Borrelia Omp66 649 TCTGGCTTTGGTTTTGC Broad-Borrelia Omp66 650 TTTTGCAGTCCAGGAGT Broad-Borrelia Omp66 651 AGGAGTGAGTTCATCTA Broad-Borrelia Omp66 652 CATCTATGTCAAATCTG Broad-Borrelia Omp66 653 AATCTGAACTCACTCTT Broad-Borrelia Omp66 654 ACTCTTGTTTTCAAAT Broad-Borrelia Omp66 655 GGAATAAGACCTTTTTT Broad-Borrelia Omp66 656 TCAGAGATTATCTTTAG Broad-Borrelia Omp66 657 AAGAATTACTCCCACTA Broad-Borrelia Omp66 658 CCACTAATTGCCCATGA Broad-Borrelia Omp66 659 AAAATTAGTGGTTCTAT Broad-Borrelia Omp66 660 TTGATCTGTGTTTGCTT Broad-Borrelia Omp66 661 AATCGCTGGCAAATGGA Broad-Borrelia Omp66 662 GTAAGACCAATTTTGTA Broad-Borrelia Omp66 663 TTTGTAAACTAATCCAA Broad-Borrelia Omp66 664 AAGTTGGACTCACAAGA Broad-Borrelia Omp66 665 GAGTTTCCGATTCCTCC Broad-Borrelia Omp66 666 TCCTCCTGAGAGTCCAA Broad-Borrelia Omp66 667 TTATGCCTTTATAAGTT Broad-Borrelia Omp66 668 TAAGTTCCATTGTAAGG Broad-Borrelia Omp66 669 GTAAGGAGTTTCTATTT Broad-Borrelia Omp66 670 TTGTATCCAACTTGAAG Broad-Borrelia Omp66 671 CTTTGAATACCAGTCAT Broad-Borrelia Omp66 672 GTTAAAATCAAAGTTTG Broad-Borrelia Omp66 673 AGTTTGTCATCGACTCC Broad-Borrelia Omp66 674 GATTTCTCCTAAATCAA Broad-Borrelia Omp66 675 TATTTTTTTCAGGTGAT Broad-Borrelia Omp66 676 GGTGATTTAAATTTGAT Broad-Borrelia Omp66 677 TTTTGCAGTCCAGGGGT Broad-Borrelia Omp66 678 AGGGGTGAGTTCATCTA Broad-Borrelia Omp66 679 TTAATAGGTCATAAACC Broad-Borrelia Omp66 680 TAAACCCCAATTGAAGC Broad-Borrelia Omp66 681 TCAGAGATTTTCTTTAG Broad-Borrelia Omp66 682 GTATGTTGGAAGATTGA Broad-Borrelia Omp66 683 AAGAATTACTCCCTCTA Broad-Borrelia Omp66 684 CCATGATTCTTTTTCCC Broad-Borrelia Omp66 685 TTTCCCCTTCATCTTTA Broad-Borrelia Omp66 686 AAAATTAGTGGTTTGAT Broad-Borrelia Omp66 687 TTTATTTGTGTTTGCTT Broad-Borrelia Omp66 688 CTTGTAAACCAATCCAA Broad-Borrelia Omp66 689 ATCCAATGGATGCTATT Broad-Borrelia Omp66 690 TAACATTTGGACTGTTT Broad-Borrelia Omp66 691 CTGTTTTGTTCTAATAC Broad-Borrelia Omp66 692 TAAGTCCCATTGTAAGG Broad-Borrelia Omp66 693 GTAAGGAGTTTCTTTTT Broad-Borrelia Omp66 694 CTTTTTTTTTCTTTTGC Broad-Borrelia Omp66 695 TTTTGCTCCTCTTCAGA Broad-Borrelia Omp66 696 TTCAGAATCGTCATTGT Broad-Borrelia Omp66 697 TGATGATTTCTATTGCC Broad-Borrelia Omp66 698 ATTGCCTGTTCCTATTG Broad-Borrelia Omp66 699 AAACTCTATTTGTGGTA Broad-Borrelia Omp66 700 GTGGTAGCAGATTGTAT Broad-Borrelia Omp66 701 TTGTATCCAAATTGAAG Broad-Borrelia Omp66 702 CTGTTCTTTTAACTTTG Broad-Borrelia Omp66 703 ACTTTGCTTTTTGAGAA Broad-Borrelia Omp66 704 AGTCATTGGTGCGAAAC Broad-Borrelia Omp66 705 GACTCCATTTTCAGGTA Broad-Borrelia Omp66 706 CAGGTAGAAATCATATA Broad-Borrelia Omp66 707 AATTTCCCCTAAATCAA Broad-Borrelia Omp66 708 GGTGATTTGAATTTGAT Broad-Borrelia Omp66 709 TTTGATTTCTATCTGGC Broad-Borrelia Omp66 710 TTAATAGCTCATAAACC Broad-Borrelia Omp66 711 TAAACCCCAATTGAGGC Broad-Borrelia Omp66 712 TGAGGCTATCCATCCAA Broad-Borrelia Omp66 713 GGAATAATGCCTTTTTT Broad-Borrelia Omp66 714 CTTCATAAGTTGAAATC Broad-Borrelia Omp66 715 GAAATCTCAGCATTGAA Broad-Borrelia Omp66 716 ATTGAAGGTTTTAAATG Broad-Borrelia Omp66 717 TAAATGTTTTTGCAGCG Broad-Borrelia Omp66 718 GCAGCGATTTCCTGTAT Broad-Borrelia Omp66 719 CTGTATTATGTTGT Broad-Borrelia Omp66 720 TGTTGTTTGAAGATGTA Broad-Borrelia Omp66 721 GATGTAGGCTTATATAA Broad-Borrelia Omp66 722 ATATAAATTTTGTCCGT Broad-Borrelia Omp66 723 GTCCGTAGGTAATTCCA Broad-Borrelia Omp66 724 ATTCCAATTCCAATTCC Broad-Borrelia Omp66 725 AATTCCAGATTTTTTGT Broad-Borrelia Omp66 726 TTTTGTTTTGTGTGCCA Broad-Borrelia Omp66 727 GTGCCAAATATTCTTTT Broad-Borrelia Omp66 728 TCTTTTACTGTAGGAGC Broad-Borrelia Omp66 729 AGGAGCTACCTCCATTA Broad-Borrelia Omp66 730 CCATTAATTGACCATGA Broad-Borrelia Omp66 731 CCATGATTCTTTCTCAC Broad-Borrelia Omp66 732 TCTCACCTTCATCTGTA Broad-Borrelia Omp66 733 TCTGTATTCCAAGCTAA Broad-Borrelia Omp66 734 AGCTAAACCGATACCAG Broad-Borrelia Omp66 735 TACCAGTTCCTATGGAA Broad-Borrelia Omp66 736 AAAATTAGTGGTTCTTT Broad-Borrelia Omp66 737 TTTATTCCCTTTTGGAT Broad-Borrelia Omp66 738 TTGGATCGAATTGA Broad-Borrelia Omp66 739 TGAGACTTGTCATC Broad-Borrelia Omp66 740 GTCATCTGTATTTGCTT Broad-Borrelia Omp66 741 TTGCTTTTTTGGAGATG Broad-Borrelia Omp66 742 GAGATGTGTCCAAGTAT Broad-Borrelia Omp66 743 AATCGCTTGCAAATGGA Broad-Borrelia Omp66 744 AATGGATCTATGCCTAA Broad-Borrelia Omp66 745 GCCTAAATCAGAGCCTG Broad-Borrelia Omp66 746 AGCCTGTTTGTAATAAG Broad-Borrelia Omp66 747 AGTATTTCTGTTATTGA Broad-Borrelia Omp66 748 GTAAGACCAAGTTTGTA Broad-Borrelia Omp66 749 TTTGTAAGTAAATCCGA Broad-Borrelia Omp66 750 ATCCGATAGAGGTCATA Broad-Borrelia Omp66 751 GTCATAATAGATGCATT Broad-Borrelia Omp66 752 AAGTTGGGCTAACTAGA Broad-Borrelia Omp66 753 ACTAGATCTGAGTCA Broad-Borrelia Omp66 754 GTCAATAGCTGTGT Broad-Borrelia Omp66 755 CTGTGTCTTTAAGTCCA Broad-Borrelia Omp66 756 AGTCCATATGTTATTGA Broad-Borrelia Omp66 757 GAGTTTCCGATTGCTCC Broad-Borrelia Omp66 758 TGCTCCTGAGATTCCAA Broad-Borrelia Omp66 759 TTCCAAAGTTTAATTCA Broad-Borrelia Omp66 760 AATTCAAATGGAGTTTC Broad-Borrelia Omp66 761 AGTTTCTGCAATGACAT Broad-Borrelia Omp66 762 TGACATTTGAGCCGTAT Broad-Borrelia Omp66 763 CCGTATTGTTCTAGTTC Broad-Borrelia Omp66 764 TTATTGGCTTCCATTTT Broad-Borrelia Omp66 765 CATTTTACCTGAGTTCC Broad-Borrelia Omp66 766 AGTTCCATAAAGCATAC Broad-Borrelia Omp66 767 GCATACCTCGATAGGTA Broad-Borrelia Omp66 768 TAGGTATCGTTGTAAGG Broad-Borrelia Omp66 769 GTAAGGAGTTTCTTCTT Broad-Borrelia Omp66 770 TTTTTATCATCCGCTGA Broad-Borrelia Omp66 771 CGCTGAATCGTTAGCAT Broad-Borrelia Omp66 772 TAGCATTTTTTTGATGG Broad-Borrelia Omp66 773 TGATGGTTTCTGTTACC Broad-Borrelia Omp66 774 GTTACCTGTTCCTGTTG Broad-Borrelia Omp66 775 CTGTTGCTCCAATTGCA Broad-Borrelia Omp66 776 ATTGCAAGCACAAGTTC Broad-Borrelia Omp66 777 AAGTTCTAATTGTG Broad-Borrelia Omp66 778 GTGGAAGAGTGTAT Broad-Borrelia Omp66 779 GTGTATCCAAACTGAAG Broad-Borrelia Omp66 780 CTGAAGTGTGCCTATTT Broad-Borrelia Omp66 781 CTATTTTTTTTGCTGTA Broad-Borrelia Omp66 782 GCTGTACCTTTTGCAAG Broad-Borrelia Omp66 783 TGCAAGAATTGTTCTTC Broad-Borrelia Omp66 784 TTCTTCTTGTGGCGCTG Broad-Borrelia Omp66 785 GCGCTGTTATTGCTTGG Broad-Borrelia Omp66 786 GCTTGGGAAACCGTAAT Broad-Borrelia Omp66 787 CGTAATATTTACTTTGA Broad-Borrelia Omp66 788 CTTTGAATGCTAGTCAT Broad-Borrelia Omp66 789 CGAAACTAAATAATGAT Broad-Borrelia Omp66 790 AATTAGTCATTGATTCC Broad-Borrelia Omp66 791 GATTCCATTTTAAGATA Broad-Borrelia Omp66 792 AGTATGGCATCCTTTTT Broad-Borrelia Omp66 793 CTTTTTTCCTTGAGCTT Broad-Borrelia Omp66 794 GAGCTTTTATAAACAAA Broad-Borrelia Omp66 795 AACAAATCCTCTATTTT Broad-Borrelia Omp66 796 TATTTTAATGTAAGCTG Broad-Borrelia Omp66 797 GGATCATCCTTGCCTAC Broad-Borrelia Omp66 798 GCCTACTTCTTTATTTT Broad-Borrelia Omp66 799 TTTAAATCCTATCTGGC Broad-Borrelia Omp66 800 TCTGGCTTTTATTTTCC Broad-Borrelia Omp66 801 TTTTCCAAACCAGGAAT Broad-Borrelia Omp66 802 AGGAATGAGTTCATCCA Broad-Borrelia Omp66 803 CATCCATATCAAACCTA Broad-Borrelia Omp66 804 AACCTAAATTCACTGCT Broad-Borrelia Omp66 805 ACTGCTGTTTTCGAAT Broad-Borrelia Omp66 806 TTAATAGTTCATAAACC Broad-Borrelia Omp66 807 ATTGAAGGTTTGAAATG Broad-Borrelia Omp66 808 GAAATGCTTTTGCAGCG Broad-Borrelia Omp66 809 GCAGCGATTTTCTGTAT Broad-Borrelia Omp66 810 CTGTATTACTTTGT Broad-Borrelia Omp66 811 CTTTGTTTGAAGATGTA Broad-Borrelia Omp66 812 GTCCATAGGTAATACCA Broad-Borrelia Omp66 813 ATACCAATTCCAATTCC Broad-Borrelia Omp66 814 TTTTGTCTTGTGTGCCA Broad-Borrelia Omp66 815 TCTTTTACTGTAAGAGC Broad-Borrelia Omp66 816 AAGAGCTACCTCCACTA Broad-Borrelia Omp66 817 CCACTAATTGACCATGA Broad-Borrelia Omp66 818 CCATGATTCTTTTTCGC Broad-Borrelia Omp66 819 TTTCGCCTTCATCTGTA Broad-Borrelia Omp66 820 AAAATTAGTGGTTCTCT Broad-Borrelia Omp66 821 TTCTCTTTGTATCAAAT Broad-Borrelia Omp66 822 TTTATTCTCTTTCGGAT Broad-Borrelia Omp66 823 TCGGATTGAATTGA Broad-Borrelia Omp66 824 AATCACTGGCAAATGGA Broad-Borrelia Omp66 825 AATGGATCTATTCCTAA Broad-Borrelia Omp66 826 TCCTAAATCAGAGCCTG Broad-Borrelia Omp66 827 AGCCTGTTTGCAATAAG Broad-Borrelia Omp66 828 AAGTTGGACTAACTAGA Broad-Borrelia Omp66 829 ACTAGATCTGAGCCAGC Broad-Borrelia Omp66 830 GCCAGCAACAGCTGTGT Broad-Borrelia Omp66 831 CTGTGTCTTGAAGACCA Broad-Borrelia Omp66 832 AGACCATATGTTATTGA Broad-Borrelia Omp66 833 AGTTTCTGCAATGATAT Broad-Borrelia Omp66 834 TGATATCTGAGCTGTAT Broad-Borrelia Omp66 835 CTGTATTGTTCTAATTC Broad-Borrelia Omp66 836 CATTTTATCTGAGTTCC Broad-Borrelia Omp66 837 TTTTTATCATTTTCGGA Broad-Borrelia Omp66 838 TTCGGAATCATTATCAT Broad-Borrelia Omp66 839 TATCATTTTTTTGATGG Broad-Borrelia Omp66 840 TGATGGTTTCTGTTGCC Broad-Borrelia Omp66 841 GTTGCCTGTTCCTGTTG Broad-Borrelia Omp66 842 CTGAAGTGTACCTATTT Broad-Borrelia Omp66 843 GCTGTACCTCTTGCAAG Broad-Borrelia Omp66 844 TTCTTCTTGCGGCGCTG Broad-Borrelia Omp66 845 GCGCTGTCATTGCTTGG Broad-Borrelia Omp66 846 GATTCCATTTTAAGGTA Broad-Borrelia Omp66 847 TTTAAATCCTATTTGGC Broad-Borrelia Omp66 848 TTTGGCTTTTATTTTCC Broad-Borrelia Omp66 849 AGGAATGAGCTCGTCCA Broad-Borrelia Omp66 850 CGTCCATATCAAATCTA Broad-Borrelia Omp66 851 AATCTAAATTCACTGCT Broad-Borrelia Omp66 852 AATTCCAGATTTTTTGC Broad-Borrelia Omp66 853 TTTTGCCTTGTGTGCCA Broad-Borrelia Omp66 854 CCATGATTCTTTTTCAC Broad-Borrelia Omp66 855 TTTCACCTTCATCTGTA Broad-Borrelia Omp66 856 TTTATTCTCTTTTGGAT Broad-Borrelia Omp66 857 TTGGATTGAATTGA Broad-Borrelia Omp66 858 ACTAGATCTGATCCAGC Broad-Borrelia Omp66 859 TCCAGCAACAGCTGTGT Broad-Borrelia Omp66 860 GAATTTCCGATTGCTCC Broad-Borrelia Omp66 861 TGACATCTGAGCTGTAT Broad-Borrelia Omp66 862 AGTTCCATAAAGCATGC Broad-Borrelia Omp66 863 GCATGCCTTGATAGGTA Broad-Borrelia Omp66 864 TTTTTATCAGCCTCTGA Broad-Borrelia Omp66 865 CTCTGAATCGTTAGCAT Broad-Borrelia Omp66 866 GTTACCCGTTCCTGTTG Broad-Borrelia Omp66 867 CTGAAGTGAGCCTATTT Broad-Borrelia Omp66 868 TTCTTCTTGTTGCGCTG Broad-Borrelia Omp66 869 CGTAATATTTGCTTTGA Broad-Borrelia Omp66 870 AGTCATTGGCGCAAAAC Broad-Borrelia Omp66 871 AATATTGCCTACATTAA Broad-Borrelia Omp66 872 CTTTTTCCCTTGAGCTT Broad-Borrelia Omp66 873 CATCCATATCAAATCTA Broad-Borrelia Omp66 874 TAAATTCCAAGTGAAGC Broad-Borrelia Omp66 875 GGAATAAGTCCTTTTGT Broad-Borrelia Omp66 876 TTTTGTGTTGTCTTCGT Broad-Borrelia Omp66 877 CTTCGTAAGTTGAAATT Broad-Borrelia Omp66 878 GAAATTTCGGCATTAAA Broad-Borrelia Omp66 879 ATTAAAAGTTTGGAATG Broad-Borrelia Omp66 880 GGAATGATTTTGCAGCA Broad-Borrelia Omp66 881 GCAGCAATGTCCTGTAT Broad-Borrelia Omp66 882 TTTTGTTTGAAGATGTA Broad-Borrelia Omp66 883 GATGTAGGTTTATATAG Broad-Borrelia Omp66 884 TTCCATAGGTAATTCCA Broad-Borrelia Omp66 885 ATTCCAAGTCCAATTCC Broad-Borrelia Omp66 886 AATTCCGGATTTTTTGT Broad-Borrelia Omp66 887 TTTTGTCTTGTGCACCA Broad-Borrelia Omp66 888 GCACCAAATATTCTCGT Broad-Borrelia Omp66 889 TCTCGTATTATAGGAAT Broad-Borrelia Omp66 890 AGGAATTACCACCGCTA Broad-Borrelia Omp66 891 CCGCTAATTGACCATGA Broad-Borrelia Omp66 892 CCATGATTCTTGTTCAC Broad-Borrelia Omp66 893 GTTCACCGTCATCTTTA Broad-Borrelia Omp66 894 TCTTTATTCCAGGCAAA Broad-Borrelia Omp66 895 GGCAAAACCGATACCTG Broad-Borrelia Omp66 896 TACCTGTTCCTACAGAA Broad-Borrelia Omp66 897 AAAGTTGGTACTTCTTT Broad-Borrelia Omp66 898 TCAAAATTAAGCTTGTT Broad-Borrelia Omp66 899 CTTGTTTCCTGTTGGAT Broad-Borrelia Omp66 900 TTGGATCAAACTGG Broad-Borrelia Omp66 901 TGGTTTTTGTCATC Broad-Borrelia Omp66 902 GTCATCTGTATTTGCCT Broad-Borrelia Omp66 903 TTGCCTTTCTGGAGATG Broad-Borrelia Omp66 904 AATGGATCTATTCCTAC Broad-Borrelia Omp66 905 TCCTACATCAGAGCCGG Broad-Borrelia Omp66 906 AGCCGGTTTGTATTAAG Broad-Borrelia Omp66 907 ATTAAGAGATAGGTATT Broad-Borrelia Omp66 908 GGTATTTTTGTCATTGA Broad-Borrelia Omp66 909 CATTGATTTTTGTAAGA Broad-Borrelia Omp66 910 GTAAGACCAAGCTTATA Broad-Borrelia Omp66 911 CTTATAAGTAAATCCAA Broad-Borrelia Omp66 912 ATCCAATAGAGGCCATT Broad-Borrelia Omp66 913 GCCATTATAGATGCGTT Broad-Borrelia Omp66 914 TGCGTTTGATAAAGTTG Broad-Borrelia Omp66 915 ACAAGATCTGAGCCTAT Broad-Borrelia Omp66 916 GCCTATAACAGATTTGT Broad-Borrelia Omp66 917 ATTTGTCTTTAAGTCCG Broad-Borrelia Omp66 918 AGTCCGTATGTTATTGA Broad-Borrelia Omp66 919 TGCTCCTGAGAGTCCAA Broad-Borrelia Omp66 920 GTCCAAAATGTAATTCA Broad-Borrelia Omp66 921 AATTCAAATGGAATTTC Broad-Borrelia Omp66 922 AATTTCTGCAATGACAT Broad-Borrelia Omp66 923 CTGTATTGTGCTAGTTC Broad-Borrelia Omp66 924 TAGTTCATTTTTCATTG Broad-Borrelia Omp66 925 TCATTGGTGTCCATTTC Broad-Borrelia Omp66 926 CATTTCACTTTTGTTCC Broad-Borrelia Omp66 927 TGTTCCGTAAAGTATAC Broad-Borrelia Omp66 928 GTATACCTTGATATGTA Broad-Borrelia Omp66 929 TATGTATTGTTGTAAGG Broad-Borrelia Omp66 930 GTAAGGAGTTTCGTCTT Broad-Borrelia Omp66 931 CGTCTTTTTTCTTTTTA Broad-Borrelia Omp66 932 TTTTTATCTTCTTCGGA Broad-Borrelia Omp66 933 TTCGGAATCGTTAGCAT Broad-Borrelia Omp66 934 TAGCATTTTTTTTGTAG Broad-Borrelia Omp66 935 TTGTAGTTTCTGTTTCC Broad-Borrelia Omp66 936 GTTTCCTGTTCCTGTTG Broad-Borrelia Omp66 937 CTGTTGCCCCAATTGCA Broad-Borrelia Omp66 938 ATTGCAAGCAAAAGTTC Broad-Borrelia Omp66 939 GTGGAAAAGTATAT Broad-Borrelia Omp66 940 GTATATCCAAACTGAAG Broad-Borrelia Omp66 941 CTGAAGTGTTCCTATTG Broad-Borrelia Omp66 942 CTATTGTTTTTGCTGTA Broad-Borrelia Omp66 943 GCTGTACTTCTTGCAAG Broad-Borrelia Omp66 944 TGCAAGGATTGTTCTTC Broad-Borrelia Omp66 945 TTCTTCTTGTAGCGCAA Broad-Borrelia Omp66 946 GCGCAATCATTGCTTGG Broad-Borrelia Omp66 947 GCTTGGAAAACCGTAAT Broad-Borrelia Omp66 948 CGTAATATTCGCTTTGC Broad-Borrelia Omp66 949 CTTTGCATACTAGTCAT Broad-Borrelia Omp66 950 GTTAAAATCAAAATCAG Broad-Borrelia Omp66 951 AATCAGTCATTGATTCC Broad-Borrelia Omp66 952 CATATATGTTGATTTGT Broad-Borrelia Omp66 953 ATTTGTGTTGTAATGCT Broad-Borrelia Omp66 954 AATGCTTCCTACATCGA Broad-Borrelia Omp66 955 CATCGAGTTTGAGTATG Broad-Borrelia Omp66 956 CTTTTTGCCCTGAGCCT Broad-Borrelia Omp66 957 GAGCCTTTAACACGAGG Broad-Borrelia Omp66 958 ACGAGGTCCTCTACTTT Broad-Borrelia Omp66 959 TACTTTGATGTAAGCTG Broad-Borrelia Omp66 960 GGATCATGTTTTCCTAT Broad-Borrelia Omp66 961 TCCTATTTCATTATGTG Broad-Borrelia Omp66 962 TATGTGTTTCATATGGT Broad-Borrelia Omp66 963 TATGGTACAAATTTAAG Broad-Borrelia Omp66 964 TTTAAGTCCTAGCTGGC Broad-Borrelia Omp66 965 GCTGGCTTTTATTTTCC Broad-Borrelia Omp66 966 AGGAATGAGCTCATCCA Broad-Borrelia Omp66 967 AATCTAAATTCGCTACT Broad-Borrelia Omp66 968 GCTACTATTTTCGAAT Broad-Borrelia Omp66 969 GGAATGATTTTGCAGCT Broad-Borrelia Omp66 970 GCAGCTATGTCCTGTAT Broad-Borrelia Omp66 971 GGAATAATTCCTTTTGT Broad-Borrelia Omp66 972 GGAATGATTTTGCAGCG Broad-Borrelia Omp66 973 GCAGCGATATCCTGTAT Broad-Borrelia Omp66 974 ATATAGGTTTTTTCCAT Broad-Borrelia Omp66 975 AATTCCTGATTTTTTGT Broad-Borrelia Omp66 976 TTTTGTTTTGTGTTCCA Broad-Borrelia Omp66 977 GTTCCAAATATTCTTGT Broad-Borrelia Omp66 978 TCTTGTATTATAGGAAT Broad-Borrelia Omp66 979 AGGAATTATCACCGCTA Broad-Borrelia Omp66 980 GTTCACCGTCATCTGTA Broad-Borrelia Omp66 981 AGCTAAACCGATACCTG Broad-Borrelia Omp66 982 AAAATTGGTACTTCTTT Broad-Borrelia Omp66 983 TTTGTTTTCTGTTGGAT Broad-Borrelia Omp66 984 TTGGATTAAATTGG Broad-Borrelia Omp66 985 TGGTCTTTGTCGTC Broad-Borrelia Omp66 986 GTCGTCTGTATTTGCCT Broad-Borrelia Omp66 987 GTAAGACCAAGCTTGTA Broad-Borrelia Omp66 988 CTTGTACGTAAATCCAA Broad-Borrelia Omp66 989 ACAAGATCTGAGCCTAC Broad-Borrelia Omp66 990 GCCTACAACAGATTTGT Broad-Borrelia Omp66 991 GTCCAAAGTGTAATTCA Broad-Borrelia Omp66 992 TTGTAGTTTCTGTTCCC Broad-Borrelia Omp66 993 GTTCCCTGTTCCTGTTG Broad-Borrelia Omp66 994 GTGGAAAAGTGTAT Broad-Borrelia Omp66 995 GCGCAATCATCGCTTGG Broad-Borrelia Omp66 996 CGTAATATTCGCTTTGA Broad-Borrelia Omp66 997 CTTTGAATACTAGTCAT Broad-Borrelia Omp66 998 AATCAGTCATTGATTCT Broad-Borrelia Omp66 999 GATTCTATTTTGAGGTA Broad-Borrelia Omp66 1000 CTTTTTGCCCTTAGCTT Broad-Borrelia Omp66 1001 TAGCTTTTAACACGAGG Broad-Borrelia Omp66 1002 GGATCATGCTTTCCTAT Broad-Borrelia Omp66 1003 TCCTATTTCGTTATGTG Broad-Borrelia Omp66 1004 TTTAATTCCTAGCTGGC Broad-Borrelia Omp66 1005 AGGAATGGACTCATCCA Broad-Borrelia Omp66 1006 CATCCATGTCAAATCTA Broad-Borrelia Omp66 1007 TTTTGTGTTATCTTCGT Broad-Borrelia Omp66 1008 GCAGCGATATTCTGTAT Broad-Borrelia Omp66 1009 TCAAATTGAAGTTTGTT Broad-Borrelia Omp66 1010 GAGTTTCCGATTGCCCC Broad-Borrelia Omp66 1011 TGCCCCTGAGAGTCCAA Broad-Borrelia Omp66 1012 TACTTGGACACATCTCC Broad-Borrelia Omp66 1013 TTCCACAATTAGAACTT Broad-Borrelia Omp66

TABLE 24 Seq. Gene/ ID DIANA Sequence Target Plasmid 1014 ATTTTTAACCACATCTG B. burgdorferi Omp66 1015 GGATGCCAACATTTGGA B. burgdorferi Omp66 1016 TTTGGATTTGAAAACAC B. burgdorferi Omp66 1017 GATATGGACGAGCTTGT B. burgdorferi Omp66 1018 AAGATCTTGCACTAAAA B. burgdorferi Omp66 1019 GGGAGATATTACAGCCC B. burgdorferi Omp66 1020 TTTAGTTTTGCACCTAT B. burgdorferi Omp66 1021 ACCTATGACTGGATTTA B. burgdorferi Omp66 1022 AACAATTCAGCTGGGAT B. burgdorferi Omp66 1023 CTCCCAAAACTCGACCT B. burgdorferi Omp66 1024 TCCATACAATAAAACAT B. burgdorferi Omp66 1025 AAACATATCAAGGAATC B. burgdorferi Omp66 1026 GGAATCCTTTATGGAAT B. burgdorferi Omp66 1027 CAACATGGAAACCAATA B. burgdorferi Omp66 1028 ACTAAATCTGTAATTGC B. burgdorferi Omp66 1029 CTTGTCAGGAGCCTATG B. burgdorferi Omp66 1030 CCTATGGAAACGAGACA B. burgdorferi Omp66 1031 GAGACATTCAATAATTC B. burgdorferi Omp66 1032 GCAACGATTTATTGAGC B. burgdorferi Omp66 1033 TTGAGCCCAACTTTATC B. burgdorferi Omp66 1034 GCATCTTTTGGAGCTAA B. burgdorferi Omp66 1035 AGCTAAATATAAGCTTG B. burgdorferi Omp66 1036 CGATAAAAATACCTATC B. burgdorferi Omp66 1037 CCTATCTTATTTTGCAA B. burgdorferi Omp66 1038 TTGCAAATGGGAACTGA B. burgdorferi Omp66 1039 AACTGATTTTGGAATAG B. burgdorferi Omp66 1040 TCAAAAGCAGCGAATTT B. burgdorferi Omp66 1041 AAGAAACACCCTCAGAT B. burgdorferi Omp66 1042 TCAGATCCTAACAAAAA B. burgdorferi Omp66 1043 GAAATATTTGATCCAAA B. burgdorferi Omp66 1044 ATTTCAGCAAAAACACA B. burgdorferi Omp66 1045 AACACAGAATTGGGCAT B. burgdorferi Omp66 1046 GCAAGTATAGGTTTTGC B. burgdorferi Omp66 1047 TTTTGCTTGGAATAAAG B. burgdorferi Omp66 1048 ATCCTGGGCGATTAAAG B. burgdorferi Omp66 1049 TACAAGACTCTTTGGAG B. burgdorferi Omp66 1050 TGGAGTTGCATTGGGAA B. burgdorferi Omp66 1051 ACTATCCTACAACAATT B. burgdorferi Omp66 1052 ACAATTTCAAGCACCAC B. burgdorferi Omp66 1053 CTTATATCTTGATTATG B. burgdorferi Omp66 1054 CCAACATTTGGATTTGA B. burgdorferi Omp66 1055 TTTTGCACCTATGACTG B. burgdorferi Omp66 1056 CAATAAAACATATCAAG B. burgdorferi Omp66 1057 ATCAAGGAATCCTTTAT B. burgdorferi Omp66 1058 CAGGAGCCTATGGAAAC B. burgdorferi Omp66 1059 ATTTATTGAGCCCAACT B. burgdorferi Omp66 1060 TTTTGGAGCTAAATATA B. burgdorferi Omp66 1061 AAATACCTATCTTATTT B. burgdorferi Omp66 1062 GTATAGGTTTTGCTTG B. burgdorferi Omp66 1063 ATGGGAACTGATTTTGG B. burgdorferi Omp66 1064 CACCCTCAGATCCTAAC B. burgdorferi Omp66 1065 GCAAAAACACAGAATTG B. burgdorferi Omp66 1066 TATAGGTTTTGCTTGGA B. burgdorferi Omp66 1067 GACTCTTTGGAGTTGCAT B. burgdorferi Omp66 1068 CCTACAACAATTTCAAG B. burgdorferi Omp66 1069 CAAAAATAAACGATAAA B. burgdorferi Omp66 1070 ACAAAAAAGCTGAAATA B. burgdorferi Omp66 1071 AAACACAGAATTGGGCA B. burgdorferi Omp66 1072 GCACCACTGAAAACAA B. burgdorferi Omp66 1073 CAATCAAACTGAACAA B. burgdorferi Omp66 1074 CTTATTTTGCAAATGGG B. burgdorferi Omp66

TABLE 25 Seq. Gene/ ID DIANA Sequence Target Plasmid 1075 CATTAAAGTGGAAGATC B. afzelli Omp66 1076 GCGATCCATTTAAAATT B. afzelli Omp66 1077 CAGCCCAAATTAATATA B. afzelli Omp66 1078 AAGAATCTTTATTTAGC B. afzelli Omp66 1079 TTTAGCTTTGCACCCAT B. afzelli Omp66 1080 ACCCATGACCGGATTCA B. afzelli Omp66 1081 GACAAAGACGCTCCATA B. afzelli Omp66 1082 GGAATACTTTATGGGGT B. afzelli Omp66 1083 TGGGGTTCAAGCAACAT B. afzelli Omp66 1084 CTTATCAGGAGCTTATG B. afzelli Omp66 1085 ATCTGTAGTTGGTAACG B. afzelli Omp66 1086 AAGGAATATCCACAGAT B. afzelli Omp66 1087 ACAGATCCTAGTAAAAA B. afzelli Omp66 1088 AAGATATATTTGATCCA B. afzelli Omp66 1089 AATACAGAACTAGGCAT B. afzelli Omp66 1090 AGGCATTGCATTTTCAA B. afzelli Omp66 1091 GCAAGCATAGGGCTTGC B. afzelli Omp66 1092 GCTTGCTTGGAATAAAG B. afzelli Omp66 1093 ATCTTGGAAAGTTAAAG B. afzelli Omp66 1094 CGGGGTTGCATTAGGAA B. afzelli Omp66 1095 TATGGGCAAAATCTTTA B. afzelli Omp66 1096 TCTTTACAGATCTAAAG B. afzelli Omp66 1097 ACTATCCTACATCAACC B. afzelli Omp66 1098 TCAACCTTAAGTGCTAA B. afzelli Omp66 1099 TGCTAATGAGAACAATC B. afzelli Omp66 1100 ACTGGACAAAGTTCAAC B. afzelli Omp66 1101 TTCAACAGGCACACAAG B. afzelli Omp66 1102 CACAAGCCATAACACCT B. afzelli Omp66 1103 ACACCTAATCTAACATT B. afzelli Omp66 1104 ACGCAATGAAACTAGGC B. afzelli Omp66 1105 CTAGGCATAGCTTTATA B. afzelli Omp66 1106 AAGCATATGTAGTACCA B. afzelli Omp66 1107 GTACCATATATTGGAGC B. afzelli Omp66 1108 ATCAAGCGATGCTACAA B. afzelli Omp66 1109 CTAATAAAAACGCAAAT B. afzelli Omp66 1110 GCAAATAATGCTGCTAT B. afzelli Omp66 1111 TGCTATTGGCAGTGCTT B. afzelli Omp66 1112 TCTTTATTTAGCTTTGCA B. afzelli Omp66 1113 CTTTGCACCCATGACCG B. afzelli Omp66 1114 CTTTATGGGGTTCAAGC B. afzelli Omp66 1115 TATCCACAGATCCTAGT B. afzelli Omp66 1116 GAACTAGGCATTGCATT B. afzelli Omp66 1117 CATAGGGCTTGCTTGGA B. afzelli Omp66 1118 CAAAATCTTTACAGATC B. afzelli Omp66 1119 CCTACATCAACCTTAAG B. afzelli Omp66 1120 CTTAAGTGCTAATGAGA B. afzelli Omp66 1121 CAAAGTTCAACAGGCAC B. afzelli Omp66 1122 CAGGCACACAAGCCATA B. afzelli Omp66 1123 GCCATAACACCTAATCT B. afzelli Omp66 1124 TGAAACTAGGCATAGCT B. afzelli Omp66 1125 ATGTAGTACCATATATT B. afzelli Omp66 1126 AAAACGCAAATAATGCT B. afzelli Omp66 1127 AATGCTGCTATTGGCAG B. afzelli Omp66 1128 GCTATGGGCAAAATCT B. afzelli Omp66

TABLE 26  Seq. Gene/ ID DIANA Sequence Target Plasmid 1129 GGATGCCGACATTCGGA B. mayonii Omp66 1130 TTCGGATTTGAAAACAC B. mayonii Omp66 1131 AGGAGATATAACAGCCC B. mayonii Omp66 1132 TTTAGTTTTGCGCCTAT B. mayonii Omp66 1133 GCCTATGACTGGATTTA B. mayonii Omp66 1134 GAGGTACTTCTAAAAAG B. mayonii Omp66 1135 AACAATTCAATTGGGAT B. mayonii Omp66 1136 CTCCCACAACTCGACCT B. mayonii Omp66 1137 ACTGAATCTGTAGTTGC B. mayonii Omp66 1138 AGTTGCAGAAATACCTT B. mayonii Omp66 1139 CTTGTCAGGAGCTTATG B. mayonii Omp66 1140 CTTATGGAAACGAAACA B. mayonii Omp66 1141 GCAACGATTTATTGAGT B. mayonii Omp66 1142 GCATCTTTTGGGGCTCA B. mayonii Omp66 1143 GGCTCAATATAAGCTCG B. mayonii Omp66 1144 AGCTCGGATTAACAAAA B. mayonii Omp66 1145 TTACAAATGGGCACTGA B. mayonii Omp66 1146 CACTGATTTAGGAATAG B. mayonii Omp66 1147 TTGGACACATATCAAAA B. mayonii Omp66 1148 TCAAAAGCAGCAAATTT B. mayonii Omp66 1149 AAGGAACATCCTCAGAT B. mayonii Omp66 1150 TCAGATCCTAGCAAAAA B. mayonii Omp66 1151 AGAATATATTTGATCCA B. mayonii Omp66 1152 GATCCAAATGGAAATGC B. mayonii Omp66 1153 AAATGCTCTTAATTTCA B. mayonii Omp66 1154 AATACAGAATTGGGCAT B. mayonii Omp66 1155 ATTTCAGCAAAAATACA B. mayonii Omp66 1156 GCAAGTATAGGACTTGC B. mayonii Omp66 1157 ACTTGCTTGGAATAAAG B. mayonii Omp66 1158 ATCCTGGAAAGTTAAAG B. mayonii Omp66 1159 GATTCCTACAGCACAAG B. mayonii Omp66 1160 CACAAGGTTATTTGGAG B. mayonii Omp66 1161 TGGGATTGCATTAGGAA B. mayonii Omp66 1162 ACTATCCTACAGTAATT B. mayonii Omp66 1163 GTAATTTCAAGCACTAA B. mayonii Omp66 1164 CACTAATGAAAATAATC B. mayonii Omp66 1165 GCTGGACAAAGTTCAAT B. mayonii Omp66 1166 TTCAATAAGCAAACAAG B. mayonii Omp66 1167 AACAAGCTACAATACCT B. mayonii Omp66 1168 ATACCTAATCTGACATT B. mayonii Omp66 1169 GACATTTGAAGACGCAA B. mayonii Omp66 1170 ACGCAATGAAGCTCGGT B. mayonii Omp66 1171 CTCGGTTTGGCTTTATA B. mayonii Omp66 1172 CCAATAACATCTATTTC B. mayonii Omp66 1173 TATTTCAACAGAAGCAT B. mayonii Omp66 1174 GTACCTTATATTGGAGC B. mayonii Omp66 1175 TTTTAGGGCCTTCTAAC B. mayonii Omp66 1176 TCTAACAAACTCTCAAG B. mayonii Omp66 1177 TATAGAACTTGCCAATA B. mayonii Omp66 1178 CCAATAAAAACGCAAAT B. mayonii Omp66 1179 GCAAATAATGCAGCTAT B. mayonii Omp66 1180 AGCTATTGGCAGTGCTT B. mayonii Omp66 1181 CCGACATTCGGATTTGA B. mayonii Omp66 1182 TTTTGCGCCTATGACTG B. mayonii Omp66 1183 TGACTGGAGGTACTTCT B. mayonii Omp66 1184 TCTGTAGTTGCAGAAAT B. mayonii Omp66 1185 CAGGAGCTTATGGAAAC B. mayonii Omp66 1186 TTTTGGGGCTCAATATA B. mayonii Omp66 1187 ATATAAGCTCGGATTAA B. mayonii Omp66 1188 ATGGGCACTGATTTAGG B. mayonii Omp66 1189 CACATATCAAAAGCAGC B. mayonii Omp66 1190 CATCCTCAGATCCTAGC B. mayonii Omp66 1191 TATTTGATCCAAATGGA B. mayonii Omp66 1192 AATGGAAATGCTCTTAA B. mayonii Omp66 1193 CTCTTAATTTCAGCAAA B. mayonii Omp66 1194 GCAAAAATACAGAATTG B. mayonii Omp66 1195 TATAGGACTTGCTTGGA B. mayonii Omp66 1196 CTACAGCACAAGGTTAT B. mayonii Omp66 1197 CTACAGTAATTTCAAGC B. mayonii Omp66 1198 TTCAAGCACTAATGAAA B. mayonii Omp66 1199 ACAAAGTTCAATAAGCA B. mayonii Omp66 1200 TAAGCAAACAAGCTACA B. mayonii Omp66 1201 CTACAATACCTAATCTG B. mayonii Omp66 1202 TAATCTGACATTTGAAG B. mayonii Omp66 1203 TGAAGACGCAATGAAGC B. mayonii Omp66 1204 TGAAGCTCGGTTTGGCT B. mayonii Omp66 1205 ACATCTATTTCAACAGA B. mayonii Omp66 1206 GGCCTTCTAACAAACTC B. mayonii Omp66 1207 ACTTGCCAATAAAAACG B. mayonii Omp66 1208 AAAACGCAAATAATGCA B. mayonii Omp66 1209 AATGCAGCTATTGGCAG B. mayonii Omp66 1210 CAAATGGAAATGCTCT B. mayonii Omp66 1211 AATTCCAATAACATCTA B. mayonii Omp66 1212 CAAGCACTAATGAAAAT B. mayonii Omp66 1213 GCACAAGGTTATTTGGA B. mayonii Omp66 1214 TTACAAATGGGCACTGA B. mayonii Omp66 1215 GTATAGGACTTGCTTGG B. mayonii Omp66

TABLE 27  Seq. Gene/ ID DIANA Sequence Target Plasmid 1216 GTGATCCATTTAAAATT B. garinii Omp66 1217 CAGCCCAAATTAATATG B. garinii Omp66 1218 TTTAGTTTTGCGCCCAT B. garinii Omp66 1219 GCCCATGACTGGATTCA B. garinii Omp66 1220 CTTATCGGGAGCTTATG B. garinii Omp66 1221 ATCTTTAGTTGGTAACG B. garinii Omp66 1222 AAGGAATATCCGTAGAT B. garinii Omp66 1223 GTAGATCCTATTAAAAA B. garinii Omp66 1224 AAGATATACTTGATCCA B. garinii Omp66 1225 GATCCAAATAGCAATGC B. garinii Omp66 1226 AATACAGAGCTGGGCAT B. garinii Omp66 1227 GCAAGCATAGGGCTTCT B. garinii Omp66 1228 GCTTCTTTGGAATAAAG B. garinii Omp66 1229 ATCTTGGAAGGTTAAGG B. garinii Omp66 1230 TTAAGGGAGCTGATTCC B. garinii Omp66 1231 TGGGGTTGCATTAGGAA B. garinii Omp66 1232 TATGGACAAAATCTTTA B. garinii Omp66 1233 TCTTTATAGATCCAAAG B. garinii Omp66 1234 AAAAACCATATCCGAAA B. garinii Omp66 1235 CCGAAAATGCATTTCAA B. garinii Omp66 1236 ACTATCCCACAACAACA B. garinii Omp66 1237 ACAACAAGCTCAGCTTC B. garinii Omp66 1238 AGCTTCTGATGCAAACA B. garinii Omp66 1239 CAAACAATCAAGCCGGA B. garinii Omp66 1240 GCCGGACAAAGTTCAGA B. garinii Omp66 1241 TTCAGAAAGCACACAAG B. garinii Omp66 1242 CACAAGCTATAACCCCT B. garinii Omp66 1243 ACCCCTAATCTAACATT B. garinii Omp66 1244 ACGCAATGAAACTTGGT B. garinii Omp66 1245 CTTGGTATAGCTTTATA B. garinii Omp66 1246 AAGCATATGTAGTACCC B. garinii Omp66 1247 GTACCCTATATTGGGGC B. garinii Omp66 1248 TGGGGCATACCTTTTAG B. garinii Omp66 1249 TTTTAGGGCCTTCTAAT B. garinii Omp66 1250 TCTAATAAAATCTCAAG B. garinii Omp66 1251 TATTTAAAGACAGGACT B. garinii Omp66 1252 AGGACTTAGTCTTGAAA B. garinii Omp66 1253 AACAATTTCTCTTGGCT B. garinii Omp66 1254 TTGGCTGGGATTCAAAT B. garinii Omp66 1255 ACAAATAATGCTGCCAT B. garinii Omp66 1256 TGCCATTGGTAGTGCTT B. garinii Omp66 1257 TTTTGCGCCCATGACTG B. garinii Omp66 1258 TATCCGTAGATCCTATT B. garinii Omp66 1259 TACTTGATCCAAATAGC B. garinii Omp66 1260 CATAGGGCTTCTTTGGA B. garinii Omp66 1261 GAAGGTTAAGGGAGCTG B. garinii Omp66 1262 CAAAATCTTTATAGATC B. garinii Omp66 1263 CCATATCCGAAAATGCA B. garinii Omp66 1264 CCCACAACAACAAGCTC B. garinii Omp66 1265 AGCTCAGCTTCTGATGC B. garinii Omp66 1266 CTGATGCAAACAATCAA B. garinii Omp66 1267 ATCAAGCCGGACAAAGT B. garinii Omp66 1268 CAAAGTTCAGAAAGCAC B. garinii Omp66 1269 AAGCACACAAGCTATAA B. garinii Omp66 1270 GCTATAACCCCTAATCT B. garinii Omp66 1271 TGAAACTTGGTATAGCT B. garinii Omp66 1272 ATGTAGTACCCTATATT B. garinii Omp66 1273 CTATATTGGGGCATACC B. garinii Omp66 1274 CATACCTTTTAGGGCCT B. garinii Omp66 1275 GGCCTTCTAATAAAATC B. garinii Omp66 1276 AAGACAGGACTTAGTCT B. garinii Omp66 1277 TTCTCTTGGCTGGGATT B. garinii Omp66 1278 AATGCTGCCATTGGTAG B. garinii Omp66 1279 GATCCTATTAAAAAAGC B. garinii Omp66 1280 TCTTTATAGATCCAAAG B. garinii Omp66 1281 AGCTCAGCTGCTGATGC B. garinii Omp66 1282 GGTATAGCTTTATATCT B. garinii Omp66

TABLE 28 Seq. Gene/ ID DIANA Sequence Target Plasmid 1283 GCTATGGGCAAAATCT B. spielmanii Omp66 1284 GAATAAAAACGACGGTG B. spielmanii Omp66 1285 TATCCTTAGATCCTAGT B. spielmanii Omp66

TABLE 29 Seq. Gene/ ID DIANA Sequence Target Plasmid 1286 TAACAAGCCCAAGCGCT B. bissettii Omp66 1287 GTTCAACAACACAAGCT B. bissettii Omp66 1288 GCGCTAATTCAGACAAT B. bissettii Omp66 1289 GGGAATTGCATTCTCAA B. bissettii Omp66 1290 TTGCACTGGGAATAAGT B. bissettii Omp66

TABLE 30 Seq. Gene/ ID DIANA Sequence Target Plasmid 1291 GATATCTATCTTATTTT B. bavariensis Omp66 1292 GCAACAAACTCAGCTAC B. bavariensis Omp66 1293 CAGATCCAAGGATACAG B. bavariensis Omp66 1294 CTTATGAATGGAATAGG B. bavariensis Omp66 1295 TGGAATAGATCCTTTCG B. bavariensis Omp66

TABLE 31 Seq. Gene/ ID DIANA Sequence Target Plasmid 1296 TATAAACTTGGATTAAC B. valaisiana Omp66 1297 CGTATGTAATACCCTAT B. valaisiana Omp66 1298 CAAGTGCTAATGCAAAC B. valaisiana Omp66 1299 GCATAGGCCTTATTTGG B. valaisiana Omp66 1300 TAAGGGGTCTGATTCAT B. valaisiana Omp66 1301 AAAGGACTTATAAATGG B. valaisiana Omp66 1302 TCCTAGCAAAAAAGGCG B. valaisiana Omp66 1303 GGAAAAAATTCAACAAG B. valaisiana Omp66

TABLE 32 Seq. Gene/ ID DIANA Sequence Target Plasmid 1304 AAAAAGGGGCTTATGAA B. spielmanii & Omp66 B. afzelii 1305 CCTACATCAACCTTAAG B. spielmanii & Omp66 B. afzelii 1306 GAAACTAGGCATAGCTT B. spielmanii & Omp66 B. afzelii 1307 CCTTAAGTGCTAATGAG B. spielmanii & Omp66 B. afzelii

TABLE 33 Seq. Gene/ ID DIANA Sequence Target Plasmid 1310 TTGCTTTTTTAGATATG B. miyamotoi Omp66 1311 AATGGATCTATTCCAAG B. miyamotoi Omp66 1312 TCCAAGGTCATAACCTG B. miyamotoi Omp66 1313 AACCTGTTTTTAGTATT B. miyamotoi Omp66 1314 AGTATTAGGTAAGTATT B. miyamotoi Omp66 1315 GCTATTATGGATGCATT B. miyamotoi Omp66 1316 ACAAGATCTGAGTTAGT B. miyamotoi Omp66 1317 GTTAGTAATAGCTGTAT B. miyamotoi Omp66 1318 CTGTATTTTGTACAGTA B. miyamotoi Omp66 1319 ACAGTATATGTTGATGA B. miyamotoi Omp66 1320 TGATGATGAATGATTAA B. miyamotoi Omp66 1321 AATTCAAAAGGGGTTTC B. miyamotoi Omp66 1322 GGTTTCTACAATAACAT B. miyamotoi Omp66 1323 GTATTGGTTTCCATTTT B. miyamotoi Omp66 1324 CATTTTAATTGAGTTCC B. miyamotoi Omp66 1325 AGTTCCGTAAATTATGC B. miyamotoi Omp66 1326 CATTGTTTTTTTGATGA B. miyamotoi Omp66 1327 CTATTGCTCCAATTGCA B. miyamotoi Omp66 1328 ATTGCAATCAAAAACTC B. miyamotoi Omp66 1329 TTGAAGTGTACCTGTTC B. miyamotoi Omp66 1330 TGAGAAAATTTTTTGAC B. miyamotoi Omp66 1331 TTTGACCTATATCTCTG B. miyamotoi Omp66 1332 TCTCTGTTCTTACTTGG B. miyamotoi Omp66 1333 ACTTGGAAAACCGTAGT B. miyamotoi Omp66 1334 CGTAGTATTTACTTTGA B. miyamotoi Omp66 1335 CGAAACTGAATAATGAT B. miyamotoi Omp66 1336 AATATGGCATCTTTTGT B. miyamotoi Omp66 1337 TTTTGTTCCTTGAGCTT B. miyamotoi Omp66 1338 GAGCTTTGAAGCCCACA B. miyamotoi Omp66 1339 CCCACATTTTCTATTTT B. miyamotoi Omp66 1340 TATTTTGATGTAAGCTG B. miyamotoi Omp66 1341 GGGTCGTCTTTTCCTAT B. miyamotoi Omp66 1342 TCTGGCTTTGGTTTTGC B. miyamotoi Omp66 1343 CATCTATGTCAAATCTG B. miyamotoi Omp66 1344 AATCTGAACTCACTCTT B. miyamotoi Omp66 1345 ACTCTTGTTTTCAAAT B. miyamotoi Omp66 1346 ATTCAAAAGGGGTTTC B. miyamotoi Omp66 1347 GTTCCGTAAATTATGC B. miyamotoi Omp66 1348 TGATGATGAATGATTA B. miyamotoi Omp66 1349 GAAAGCTCTGCATTGAG B. miyamotoi Omp66 1350 ATTGAGAGTTTTAAATG B. miyamotoi Omp66 1351 TAAATGATTTTTCAGAG B. miyamotoi Omp66 1352 GTCCATAGCTAATTCCA B. miyamotoi Omp66 1353 ATTCCAATTCCAACTCC B. miyamotoi Omp66 1354 TTTTATCTTGTTCACCA B. miyamotoi Omp66 1355 AGCGAAACCAATACCTA B. miyamotoi Omp66 1356 GATATGTGTCCAAATAT B. miyamotoi Omp66 1357 AAAACTCTATTTGTGGT B. miyamotoi Omp66 1358 GACCTATATCTCTGTTC B. miyamotoi Omp66

TABLE 34 Gene/ Seq. ID Primer Sequence; Forward (5′-3′) Target Plasmid 1359 ACATCTGTAGCAATATTTGCAG Broad-Borrelia Omp66 1360 ACATCTGCAGCAATATTTGCA Broad-Borrelia Omp66 1361 ACTATGACAGATTTTGACTTTAATAAAGA Broad-Borrelia Omp66 1362 ACAGATTTTGACTTTAATAAAGAGTCTTTA Broad-Borrelia Omp66 1363 ACAGATTTTGACTTTAATAAAGAATCTTTA Broad-Borrelia Omp66 1364 CCAACTTTATCAAATTCTGCAATTTT Broad-Borrelia Omp66 1365 GATCCTTTCGCAAGCGATT Broad-Borrelia Omp66 1366 TAGATCCTTTTGCAAGCGATT Broad-Borrelia Omp66 1367 TGGATAACATCTATCGGTCTTTATG Broad-Borrelia Omp66 1368 TGGATAACATCTATCGGTCTTTACG Broad-Borrelia Omp66 1369 CTTTATCAAATTCTGCAATTTTGGC Broad-Borrelia Omp66 1370 AACTTTATCAAATTCTGCAATTTTAGC Broad-Borrelia Omp66 1371 GCTATCCATCCAAGACCAGG Broad-Borrelia Omp66 1372 TTATCTTCATAAGTTGAAATCTCAGCA Broad-Borrelia Omp66 1373 TTCATAAGTTGAAAGCTCTGCA Broad-Borrelia Omp66 1374 CTTCATCTGTATTCCAAGCTAAACC Broad-Borrelia Omp66 1375 TCATCTTTATTCCAAGCGAAACC Broad-Borrelia Omp66 1376 TGATGAATGATTAAATGTTGAGTTTCC Broad-Borrelia Omp66 1377 GCTATCCATCCAAGACCAGG Broad-Borrelia Omp66 1378 CTTACAGACGAAATTAATAGAATTGCT Broad-Borrelia fla 1379 ACTTACAGATGAAATTAATAGAATTGCT Broad-Borrelia fla 1380 GAAATTAATAGAATTGCTGATCAAGC Broad-Borrelia fla 1381 GAAATTAATAGAATTGCTGATCAGGC Broad-Borrelia fla 1382 CAATATAACCAAATGCACATGTTGT Broad-Borrelia fla 1383 CAATATAACCAAATGCACATGTTAT Broad-Borrelia fla

TABLE 35 Gene/ Seq. ID Primer Sequence; Reverse (5′-3′) Target Plasmid 1384 GAAATTGTTGTAAATCTTATTAGTTTTTCAA Broad-Borrelia Omp66 1385 ATAAATTTTTGTAGCATCGCTTGA Broad-Borrelia Omp66 1386 ATAAATTTTTGTAGCATCGCTTGA Broad-Borrelia Omp66 1387 GCAAGTTCTATAATATTATTTGAATCCCA Broad-Borrelia Omp66 1388 CAAGTTCTATAATGTTATTTGAATCCCA Broad-Borrelia Omp66 1389 TTTGAATTGCAAGAAAGCACT Broad-Borrelia Omp66 1390 GCTATTTTGAATTGTAAGAAAGCACT Broad-Borrelia Omp66 1391 CATAAAGACCGATAGATGTTATCCA Broad-Borrelia Omp66 1392 CGTAAAGACCGATAGATGTTATCCA Broad-Borrelia Omp66 1393 GAGTGAGTTCAGATTTGACATAGA Broad-Borrelia Omp66 1394 GCAGTGAATTTAGATTTGATATGGA Broad-Borrelia Omp66 1395 CCTTCTCAGCTTACATCAAAATAGA Broad-Borrelia Omp66 1396 CCTTTCTCAGCTTACATTAAAATAGA Broad-Borrelia Omp66 1397 CAAAGCTCAAGGAACAAAAGATG Broad-Borrelia Omp66 1398 AAAAGCTCAAGGAAAAAAGGATG Broad-Borrelia Omp66 1399 GAAATTGTTGTAAATCTTATTAGTTTTTCAA Broad-Borrelia Omp66 1400 ATAAATTTTTGTAGCATCGCTTGA Broad-Borrelia Omp66 1401 ATAAATTTTTGTAGCATCGCTTGA Broad-Borrelia Omp66 1402 GCAAGTTCTATAATATTATTTGAATCCCA Broad-Borrelia Omp66 1403 CAAGTTCTATAATGTTATTTGAATCCCA Broad-Borrelia Omp66 1404 TTTGAATTGCAAGAAAGCACT Broad-Borrelia Omp66 1405 GCTATTTTGAATTGTAAGAAAGCACT Broad-Borrelia Omp66 1406 CATAAAGACCGATAGATGTTATCCA Broad-Borrelia Omp66 1407 CGTAAAGACCGATAGATGTTATCCA Broad-Borrelia Omp66 1408 TCTATGTCAAATCTGAACTCACTC Broad-Borrelia Omp66 1409 TCCATATCAAATCTAAATTCACTGC Broad-Borrelia Omp66 1410 TCATCTGTCATTGTAGCATCTTT Broad-Borrelia fla 1411 CATTGTAGCATCTTTTATTTGAGCA Broad-Borrelia fla 1412 AGCATCTTTTATTTGAGCATAAGATG Broad-Borrelia fla

In some embodiments, the preferred DIANA oligonucleotide is between 7-20 bases in length (i.e. 7-20 mer). In other embodiments, the preferred DIANA oligonucleotide is between 12-18 bases in length (i.e. 12-18 mer).

In some embodiments, the DIANAs provided herein comprise a sequence that is the complement, reverse, or reverse complement of a sequence described in Tables 1-33. In some embodiments, the DIANAs provided herein comprise a sequence that shares at least about 60-70% identity with a sequence described in Tables 1-33, or the complement, reverse, or reverse complement of a sequence described in Tables 1-33. In another embodiment, the DIANA has a sequence that shares at least about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity with the sequences of Tables 1-33, or the complement, reverse, or reverse complement of a sequence described in Tables 1-33. The terms “identity” or “homology” or “similarity” refer to sequence relationships between two DIANA sequences and can be determined by comparing a nucleotide position in each sequence when aligned for purposes of comparison. The term “identity” refers to the degree to which nucleic acids are the same between two sequences. The term “homology” or “similarity” refers to the relatedness of two functionally-equivalent DIANA sequences.

The DIANA sequences also include functional fragments of the sequence provided in Tables 1-33 and sequences sharing certain sequence identities with those in Tables 1-33, as described above, provided they function to specifically anneal to and identify the genomic material derived from microorganisms. In one aspect, these fragment sequences have 1, 2, 3, 4, 5, or 6 less bases at either or both ends of the original sequences in Tables 1-33. These shorter sequences are also within the scope of the present disclosure.

In addition, the DIANA sequences, including those provided in Tables 1-33 and sequences sharing certain sequence identities with those in Tables 1-33, as described above, can be incorporated into longer sequences, provided they function to specifically anneal to and identify microorganisms. In one aspect, the longer sequences have 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional bases at either or both ends of the original sequences. These longer sequences are also within the scope of the present disclosure.

In some embodiments, the PCR primers sequences provided herein comprise a sequence that shares at least about 60-70% identity with a sequence described in Tables 34 and 35. In another embodiment, the PCR primer sequences have a sequence that shares at least about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity with the sequences of Tables 34 and 35. The terms “identity” or “homology” or “similarity” refer to sequence relationships between two PCR primer sequences and can be determined by comparing a nucleotide position in each sequence when aligned for purposes of comparison. The term “identity” refers to the degree to which nucleic acids are the same between two sequences. The term “homology” or “similarity” refers to the relatedness of two functionally-equivalent PCR primer sequences.

The PCR primer sequences also include functional fragments of the sequence provided in Tables 34 and 35 and sequences sharing certain sequence identities with those in Tables 34 and 35, as described above, provided they function to specifically anneal to and identify the genomic material derived from microorganisms. In one aspect, these fragment sequences have 1, 2, 3, 4, 5, or 6 less bases at either or both ends of the original sequences in Tables 34 and 35. These shorter sequences are also within the scope of the present disclosure.

In addition, the PCR Primer sequences, including those provided in Tables 34 and 35 and sequences sharing certain sequence identities with those in Tables 34 and 35, as described above, can be incorporated into longer sequences, provided they function to specifically anneal to and identify microorganisms. In one aspect, the longer sequences have 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional bases at either or both ends of the original sequences. These longer sequences are also within the scope of the present disclosure.

In some embodiments, primarily ssDNA are targeted rather than DNA that is predominantly dsDNA. In some embodiments, ssDNA are created from dsDNA via denaturing protocols or through an asymmetric amplification process prior to DIANA tagging of the DNA molecule.

In some embodiments the DNA is entirely in duplex form. In some embodiments, the DNA is locally in duplex form.

In some embodiments, the DIANA oligonucleotide is modified to contain a one or more binding moieties. In some embodiments, the binding moiety binds the DIANA to a solid substrate. In some embodiments, the binding DIANA to a solid substrate is useful for separation or washing steps downstream. By way of example, but not by way of limitation, in some embodiments, the binding moieties include, but are not limited to, non-covalent binding moieties (e.g., such as biotin, digoxin, digitoxin) or covalent binding moieties (e.g., COOH group, NHS-ester group, malemide chemistry, and Click chemistry).

In some embodiments, the binding moiety is spaced from the DIANA probe by one or more linkers. In some embodiments, the linker is a single molecule. In some embodiments the linker is comprised of a chain of multiple individual molecules, either linear or branched, that are combined to create a single linker molecule.

In some embodiments, the DIANA comprises a linker. The linker component allows binding of the DIANA oligonucleotide to a solid-substrate and thus easily manipulate DIANAs and captured DNA. Without wishing to be bound by theory, the linker reduces steric hinderance or electrostatic repulsion effects thereby increasing the binding capacity, kinetics, dynamic range, and/or dynamics of the system. Through improved binding characteristics, the thermodynamic equilibrium is shifted resulting towards a shorter time-constant. This reduces requirements/constraints to overcome the Debye length, primarily in situations (as are quite common) when the DNA and the surface share a common charge polarity. In some embodiments, the linker is 4 atoms in length or greater. In some embodiments, the linker is 4-200 atoms in length.

In some embodiments, one or more binding moieties are used along a single linker. In some embodiments, two or more binding moieties along a single linker, wherein each linker has one or more binding moieties and wherein each binding moiety is attached to a different location along the oligonucleotide. In some embodiments, multiple binding moieties increase the surface binding kinetics and/or yield and/or efficiently, and/or strength.

In some embodiments, the DNA amplicon is first tagged with one or more DIANAs and then the hybrid complex is captured onto the solid-phase surface.

In some embodiments, the DIANA is incubated with a solid surface prior to capturing the microbial genetic material DNA.

In some embodiments, the solid-phase surface is a bead, nanoparticle, microparticle or flat substrate. In some embodiments, the solid-phase surface is further chemically modified to facilitate binding of the DIANA to it. In some embodiments, capturing a target amplicon and immobilizing it onto the solid-phase surface occurs in individuals wells or chambers on system (e.g., a plate or a chip).

As used herein, “atom” refers to a carbon atom, a nitrogen atom, an oxygen atom, or any atom capable of making two or more covalent bonds. Alternatively, in some embodiments, “atom” refers to the distance between two covalently bound atoms. By way of example, but not by way of limitation, the following structure: DIANA-(CH₂)₄₀-(binding moiety) has a linker (—(CH₂)₄₀—) with a length of 40 atoms. By way of example, but not by way of limitation, the following structure: DIANA-(CH₂)₄₀—O—(CH₂)₄₀-(binding moiety) has a linker (—(CH₂)₄₀—O—(CH₂)₄₀—) with a length of 81 atoms. By way of example, but not by way of limitation, the following structure: DIANA-(CH₂)₄₀—O—NH—(CH₂)₃₀-(binding moiety) has a linker (—(CH₂)₄₀—O—NH—(CH₂)₃₀—) with a length of 72 atoms. By way of example, but not by way of limitation, the following structure: DIANA-(CH₂)₄₀—O—N(CH₂)₃CH₃—(CH₂)₃₀-(binding moiety) has a linker (—(CH₂)₄₀—O—N(CH₂)₃CH₃—(CH₂)₃₀—) with a length of 72 atoms (the —(CH₂)₃CH₃ component branches off of the nitrogen atom and does not contribute to the length of the linker).

Microbial Genetic Material

The methods, assays, and kits disclosed herein are directed to detecting binding of DIANAs to microbial genetic material. As is used herein, “microbial genetic material” comprises polynucleotides of microorganisms. Polynucleotides includes any compound and/or substance that comprises a polymer of nucleotides (nucleotide monomer). Polynucleotides include, for example, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Exemplary polynucleotides of a microorganism include, e.g., genomic DNA, plasmid DNA, mRNA, tRNA, rRNA, and sRNA.

In some embodiments, microbial genetic material is from a bacterial cell. In some embodiments, the microbial genetic material is from a Gram-positive bacterial cell. In some embodiments, the microbial genetic material is from a Gram-negative bacterial cell. In some embodiments, the microbial genetic material is from a bacterial spirochete cell. In some embodiments, the microbial genetic material is from a fungal cell. In some embodiments, the microbial genetic material is from a bacteria of the genus Borrelia. In some embodiments, the Borrelia is of one or more of the species Borreliella afzelii, Borreliella americana, Borrelia anserine, Borrelia baltazardi, Borrelia bavariensis, Borrelia bissettiae, Borrelia brasiliensis, Borrelia burgdorferi, Borrelia californiensis, Borrelia carolinensis, Borrelia caucasica, Borrelia coriaceae, Borrelia crocidurae, Borrelia dugesii, Borrelia duttonii, Borrelia garinii, Borrelia graingeri, Borrelia harveyi, Borrelia hermsii, Borrelia hispanica, Borrelia japonica, Borrelia kurtenbachii, Borrelia lanei, Borrelia latyschewii, Borrelia lusitaniae, Borrelia mayonii, Borrelia mazzottii, Borrelia miyamotoi, Borrelia parkeri, Borrelia persica, Borrelia recurrentis, Borrelia sinica, Borrelia spielmanii, Borrelia tanukii, Borrelia theileri, Borrelia tillae, Borrelia turcica, Borrelia turdi, Borrelia turicatae, Borrelia valaisiana, Borrelia venezuelensis, and Borrelia yangtzensis.

Samples and Sample Collection

In some embodiments, the sample volume is 1 ml or greater, 5 ml or greater, 10 ml or greater, 15 ml or greater, or 20 ml or greater. In some embodiments, the sample volume is greater than 1 ml or greater than about 1 ml, greater than 5 ml or greater than about 5 ml, greater than 10 ml or greater than about 10 ml, greater than 15 ml or greater than about 15 ml, or greater than 20 ml or greater than about 20 ml. In some embodiments, the sample volume is less than or equal to about 50 mL, less than or equal to about 40 mL, less than or equal to about 30 mL, less than or equal to about 20 mL, less than or equal to about 10 mL, or less than or equal to about 5 mL. Combinations of the above-referenced ranges are also possible. For example, in some embodiments, the sample volume is between about 1 ml and about 50 ml, between about 5 ml and about 50 ml, or between about 10 ml and 20 ml. In some embodiments, larger sample volumes provide greater sensitivity to microorganisms present at low concentrations.

In some embodiments, the sample has a microbial load of less than 100 cells/sample, less than 90 cells/sample, less than 80 cells/sample, less than 70 cells/sample, less than 60 cells/sample, less than 50 cells/sample, less than 40 cells/sample, less than 30 cells/sample, less than 20 cells/sample, less than 10 cells/sample, less than 9 cells/sample, less than 8 cells/sample, less than 7 cells/sample, less than 6 cells/sample, less than 5 cells/sample, less than 4 cells/sample, less than 3 cells/sample, or less than 2 cells/sample, e.g., 1 cell/sample. The microbial load may be at least 1 cell/sample.

In some embodiments, the microbial load of the sample is less than 10,000 cells/mL of sample, less than 1,000 cells/mL of sample, less than 50 cells/mL of sample, less than 20 cells/mL of sample, less than 10 cells/mL of sample, less than 9 cells/mL of sample, less than 8 cells/mL of sample, less than 7 cells/mL of sample, less than 6 cells/mL of sample, less than 5 cells/mL of sample, less than 4 cells/mL of sample, less than 3 cells/mL of sample, less than 2 cells/mL of sample, less than 1 cells/mL of sample, less than 1 cells/10 mL of sample, less than 1 cells/20 mL of sample, less than 1 cells/50 mL of sample, or less than 1 cells/100 mL of sample. In some embodiments, the microbial load of the sample is at least cells/mL of sample, at least 0.5 cells/mL of sample, at least 1 cells/mL of sample, at least 2 cells/mL of sample, at least 5 cells/mL of sample, or at least 10 cells/mL of sample. Combinations of the above-referenced ranges are also possible.

In some embodiments, the sample is from a subject. Subjects include, but are not limited to, mammals, avians, reptiles, insects, amphibians, and fish. In some embodiments, a mammalian subject is human. In some embodiments, the subject is an adult human. In some embodiments, the subject is a child human (i.e., 2-16 years of age). In some embodiments, the subject is an infant (i.e., under 2 years of age).

In some embodiments, the subject has or is suspected of having an infection, e.g., a microbial infection. Examples of microbial infections include, for example, sepsis, pneumonia, urinary tract infections, joint infections, spinal fluid infections, etc. In some embodiments, the subject has or is suspected of having Lyme disease.

In some embodiments, the microbial cells in the sample or suspected of being in the sample, include, but are not limited to bacterial cells, e.g., of the genus Borrelia, fungal cells, viral particles, or a combination thereof.

In some embodiments, the sample comprises a bodily fluid, bodily excretion, or bodily secretion, e.g., blood, urine, saliva, stool, or sputum. In some embodiments, samples are comprised of human blood. In some embodiments, it is advantageous to utilize whole-blood or unprocessed blood as this removes the need to separate the blood into its various components, a rather laborious process.

In some embodiments, the methods described herein comprise acquiring a sample from a subject.

For assays in blood, microbial loads can be low and the potential for contaminations is a serious concern. Contaminations may come in the form of free nucleic acids or microbes (microorganisms). Contaminating microbes may come from many sources, including the patient's skin, healthcare provider, hospital equipment, etc. Provided herein are improved methods for collecting blood samples. Without wishing to be bound by theory, collecting more than one blood sample in the same draw, for example, by collecting multiple vials of blood in sequence, from the same blood-draw, or intravenous line, can allow for reduced levels of contamination in the second and additional samples because the contaminants will be contained in the first sample. This reduction in the level of contaminants likewise results in improved performance in the assays described herein. In some embodiments, acquiring a sample from a subject comprises drawing one or more vials of blood from a subject, preferably from the same blood-draw, or intravenous line. In some embodiments, the blood is drawn from a single line in the subject, e.g., a peripheral blood line or from an IV line.

In some embodiments, more than one vial of blood are drawn from the patient from the same line. Without wishing to be bound by theory, the use of two or more sample tubes for collecting the patient blood is advantageous for, among other things, reducing false-positives, increasing sensitivity, and increasing accuracy. In some embodiments, the first vial of blood is not used in the assay described herein. In some embodiments, the first vial of blood is discarded or used for alternate purposes.

In some embodiments, the vial to be used in the methods described herein contains an anticoagulant such as, for example, EDTA, which is the preferred anticoagulant to be used in the test disclosed here. In some embodiments, a volume between about 0.05-5 ml of blood is collected into the first blood vial (that which is not tested). In some embodiments, the blood volume to be tested is between about 1-50 ml.

Integrated Methods for Identifying and Evaluating Microbial Species

In some embodiments, the present technology provides a method for monitoring and/or identifying and/or characterizing microbial cells in a subject. In some embodiments, the method includes one or more of the following steps as is shown in FIG. 2 :

-   -   (i) depleting eukaryotic DNA from the sample, e.g., by         selectively lysing the eukaryotic cells, removing the free human         genetic material from the sample,     -   (ii) lysing one or more microbial cells in the sample, wherein         the lysing of one or more microbial cells releases a plurality         of microbial genetic materials,     -   (iii) isolating the plurality of microbial genetic materials,     -   (iv) amplifying the plurality of microbial genetic materials     -   (v) detecting the amplified microbial genetic material

In some embodiments prior to step (ii), the lysing of one or more microbial cells in the sample, it is beneficial to first isolate the microbial cells, e.g., by centrifugation or size exclusion chromatography. In some embodiments, is it beneficial to bring into (step (va)) contact or incubate the amplified microbial genetic materials with a plurality of duplex DNA Invading Artificial Nucleic Acids (DIANAs), and (step (vb)) detect binding of one or more DIANAs to their target microbial genetic material.

In some embodiments, all of steps (i)-(v) are performed. In some embodiments, some of steps (ii)-(v) are performed. By way of example, but not by way of limitation, in some sample matrices, it might be possible to skip step (i). For example, certain samples, e.g., urine, commonly do not require step (i) because of the low concentration of eukaryotic cells. In another non-limiting example, it might be possible to skip step (i) if the concentration of microbial cells is high enough to allow the user to utilize a smaller sample volume such that the human DNA in the eukaryotic cells is not of sufficient quantity to hinder/inhibit/reduce sensitivity/etc of downstream processes such as, but not limited to, enzymatic amplification.

The particular methods described herein are particularly suited for the ultrasensitive detection of very low levels of microorganisms, for example the detection of low microbial loads from large sample volumes, e.g., ≥5 ml. In some such embodiments, in step (i), after selectively lysing the eukaryotic cells, the eukaryotic DNA is removed from the sample by centrifugation, e.g., by centrifugation with one or more microparticles as is described below to stabilize the pellet having a low microbial load. The eukaryotic material can then be removed in the supernatant. Steps (ii)-(v) are performed as described above. In some such embodiments, in step (i), after selectively lysing the eukaryotic cells, the eukaryotic DNA is removed from the sample by the use of an anion exchanger, e.g., an anion exchange resin conjugated to a support substrate to capture/immobilize eukaryotic genomic material, allowing the separation of the sample containing microbial cells from the eukaryotic DNA. Steps (ii)-(v) are performed as described above. In some embodiments, an anion exchanger conjugated to a support substrate are known as magnetizable, electro-reactive, μ-particles or MERPs.

In some embodiments, the methods described herein are particularly suited for the ultrasensitive detection of Borrelia, which is generally present at very low levels in the blood. In some embodiments, for the ultrasensitive detection of Borrelia, the ultrasensitive detection methods described above is employed wherein, in step (i), the eukaryotic cells are lysed with a eukaryotic cell lysis reagent that specifically does not lyse Borrelia, optionally followed by centrifugation. Steps (ii)-(v) are performed as described above. In some embodiments, the Borrelia DNA amplified in step (v) is detected with one or more DIANAs comprising one or more sequences selected from the group consisting of SEQ ID NOs: 1-1358.

Particular embodiments of the methods described herein are shown in FIGS. 4-9 . For example, FIG. 4 shows (i) depleting eukaryotic DNA from the sample by selective lysis using ultrasensitive eukaryotic cell lysis solution followed by centrifugation with particles to stabilize the microbial pellet; (ii) lysing one or more microbial cells in the sample by chemical lysis, and (iii) isolating the plurality of microbial genetic materials using magnetizable, electro-reactive, μ-particles (MERPs) followed by a wash step and elution step, after which the microbial genetic material is ready for amplification and detection. FIG. shows the methods shown in FIG. 4 and additionally shows step (iv) of amplifying the plurality of microbial genetic materials. FIG. 6 shows the methods shown in FIG. 5 and additionally shows step (v) of detecting the amplified microbial genetic material via a DIANA based detection assay. FIG. 7 shows an alternative protocol to FIG. 4 in which step (i) of depleting eukaryotic DNA from the sample by selective lysis using ultrasensitive eukaryotic cell lysis solution followed by centrifugation with particles to stabilize the microbial pellet further includes the use of magnetizable, electro-reactive, μ-particles (MERPs) for a final eukaryotic DNA clean-up step before microbial cell lysis. The protocol is otherwise as that of FIG. 4 . FIG. 8 shows an alternative protocol to FIG. 4 in which step (i) of depleting eukaryotic DNA from the sample by selective lysis using ultrasensitive eukaryotic cell lysis solution followed by the incorporation of MERPs to the sample in order to capture free eukaryotic DNA. MERPs are then pelleted using a low-speed centrifugation or magnetized to separate them out from the supernatant which contains the intact microbial cells. The protocol is otherwise as that of FIG. 4 . FIG. 9 shows an alternative protocol to FIG. 4 in which step (i) of depleting eukaryotic DNA from the sample by selective lysis using ultrasensitive eukaryotic cell lysis solution followed by the incorporation of MERPs to the sample in order to capture free eukaryotic DNA. MERPs are then magnetized to separate them out from the supernatant which contains the intact microbial cells. The protocol is otherwise as that of FIG. 4 .

Depleting Eukaryotic DNA in a Sample

In some embodiments, the methods described herein comprise depleting eukaryotic DNA in a sample.

In some embodiments, the first step in the procedure is to selectively remove the human DNA from the specimen through a selective lysis process employing osmotic stress, one or more detergents, and ion exchange resins, e.g., similar to that which is described in WO 2016/044621A1 which is incorporated herein by reference.

In some embodiments, depleting eukaryotic DNA from the sample includes adding a eukaryotic cell lysis solution to the sample, wherein the eukaryotic cell lysis solution predominantly lyses eukaryotic cells as opposed to microbial cells and removing the eukaryotic DNA released by the lysis of the eukaryotic cells from the sample, wherein one or more intact microbial cells remain in the sample. For example, in some embodiments, the eukaryotic cell lysis solution predominantly lyses eukaryotic cells while leaving bacteria and/or fungi intact. Borrelia is particularly susceptible to lysis. Accordingly, in some embodiments, the eukaryotic cell lysis solution predominantly lyses eukaryotic cells while leaving Borrelia and/or additional bacteria and/or fungi intact. In some embodiments, the lysed cells are eukaryotic cells having DNA. In some embodiments, the lysed cells are white blood cells. In some embodiments, the eukaryotic DNA released by the lysis of the eukaryotic cells is further separated from the microbial cells by way of centrifugation. In some embodiments, the eukaryotic DNA released by the lysis of the eukaryotic cells is removed from the sample by size exclusion chromatography. In some embodiments, the eukaryotic DNA released by the lysis of the eukaryotic cells is removed from the sample by the use of an anion exchanger such as anion exchange microparticles followed by low-speed centrifugation. In some embodiments, the eukaryotic DNA released by the lysis of the eukaryotic cells is removed from the sample by the use of MERPs followed by size exclusion filtration. In some embodiments, the eukaryotic DNA released by the lysis of the eukaryotic cells is removed from the sample by the use of MERPs followed by magnetization. In some embodiments, the eukaryotic DNA released by the lysis of the eukaryotic cells is removed from the sample by the use of anion exchange microparticles followed allowing the anion exchange microparticles to settle. In some embodiments, the eukaryotic DNA released by the lysis of the eukaryotic cells is removed from the sample by blood filtration. In some embodiments, blood filtration is followed by capture of target pathogens on a filter.

In some embodiments, eukaryotic cells are removed from the sample in the absence of a lysis step. For example, in some embodiments, eukaryotic cells are removed from the sample by centrifugation in the absence of a lysis step. In further embodiments, eukaryotic cells are separated from microbial cells by contacting the sample with particles, e.g., magnetic particles, containing binding moieties that specifically bind the microbial cells and removing fluid containing the eukaryotic cells from the particles attached to the microbial cells.

Lysis of Eukaryotic Cells

Provided herein is a eukaryotic cell lysis solution that predominantly lyses eukaryotic cells while leaving bacteria and/or fungi intact. It will be appreciated that the eukaryotic cell lysis solution described in this section is formulated for gram positive bacteria, gram negative bacteria, and fungi generally, e.g., as may be found in a subject suspected having a variety of bloodborne infections. However, the eukaryotic cell lysis solution described in this section is not the preferred lysis solution when the presence of Borrelia is suspected, as Borrelia is especially susceptible to lysis. Eukaryotic cell lysis solutions suitable for lysing eukaryotic cells while leaving Borrelia intact are described below in the section entitled “Selective Lysis of Eukaryotic Cells while leaving Borrelia intact.”

In some embodiments, the eukaryotic cell lysis agent is a solution (hereinafter “a eukaryotic cell lysis solution”). Alternatively, in some embodiments, the eukaryotic cell lysis agent is pelleted and re-suspended in water or an aqueous buffer prior to use.

In some embodiments, the eukaryotic cell lysis solution includes one or more detergents or surfactants. In some embodiments, the detergents or surfactants are non-ionic, anionic, cationic, zwitterionic, or non-detergent sulfobetaines. Detergents and surfactants, include, but are not limited to BigCHAP, Deoxy BigCHAP, Brij 35, Brij 58P, Cymal-1, Cymal-2, Cymal-5, Cymal-6, Decyl-β-maltopyranoside, n-Dodecyl- -D-maltoside, n-Hexadecyl-β-D-maltoside, Undecyl-β-D-maltoside, Decyl-β-D-1-thiomaltopyranoside, Octyl-β-D-glucopyranoside, Decyl-β-D-1-thioglucopyranoside, Octyl-β-Dthioglucopyranoside, Digitonin, Dimethyldecylphosphine oxide (APO-10), Dodecyldimethylphosphine oxide (APO-12), IGEPAL CO-520, IGEPAL CO-630, and IGEPAL CO-720, N-Octanoyl-N-methylglucamine(MEGA-8), N-nonanoyl-N-methylglucamine(MEGA-9), N-Decanoyl-N-methylglucamine(MEGA-10), nonidet P40-substitute, Pluronic F-68, saponin, thesit, Triton X-100, Triton X-1 14, TWEEN 20, TWEEN TWEEN 80, sodium cholate, Sodium deoxycholate, sodium glycocholate, sodium taurocholate, sodium taurodeoxycholate, N-1-lauroylsarcosine, lithium dodecyl sulfate, sodium dodecyl sulfate (SDS), hexadecyltrimethyl ammonium bromide (CTAB), trimethyl(tetradecyl) ammonium bromide (TTAB), ASB-14(amidosulfobetaine-14), ASB-16(amidosulfobetaine-16), C7BzO, CHAPS, CHAPSO, EMPIGEN BB, 3-(N,N-Dimethyloctylammonio) propanesulfonate inner salt (SB3-8), 3-(decyldimethylammonio)-propanesulfonate inner salt (SB3-10), 3-(dodecyldimethylammonio)-propanesulfonate inner salt (SB3-12), 3-(N,N-dimethylmyristylammonio)-propanesulfonate(SB3-14), 3-(N,N-dimethylpalmitylammonio)-propanesulfonate(SB3-16), 3-(N,N-dimethyloctadecylammonio)-propanesulfonate(SB3-18), 3-(1-pyridinio)-1-propanesulfonate (NDSB 201), and 3-(benzyldimethylammonio) propanesulfonate (NDSB 256).

By way of example, but not by way of limitation, in some embodiments, the eukaryotic cell lysis solution has a concentration of surfactants between about 0.27% to 15% v/v, between about 0.39% to 13% v/v, between about 0.45% to 12% (v/v), or between about to 10% (v/v) of a Tween surfactant and/or between about 0.22% to 10% (v/v), between about 0.16% to 8.25% (v/v), or between about 0.44% to 6.75% (v/v) of Triton or IGEPAL. In some embodiments, the Tween surfactant is selected from the group consisting of Tween-20, Tween-40, and Tween-80. In some embodiments, the Triton is Triton X-100 or Triton X-1 14. In some embodiments, the IGEPAL is selected from the group consisting of IGEPAL CO-520, IGEPAL CO-630, and IGEPAL CO-720.

In some embodiments, the surfactants are stored individually in dry form and re-suspended prior to use.

By way of example, but not by way of limitation, in some embodiments, the eukaryotic cell lysis reaction (e.g., eukaryotic cell lysis solution combined with the sample (herein after the “mixture”)) comprise a final concentration of surfactants between about to 1% (v/v), between about 0.35% to 0.85% (v/v), between about 0.45% to 0.75% (v/v), or between about 0.55% to 0.65% (v/v) of a Tween surfactant and/or between about to 0.65% (v/v), between about 0.25% to 0.55% (v/v), or between about 0.35% to (v/v) of Triton or IGEPAL. In some embodiments, the Tween surfactant is selected from the group consisting of Tween-20, Tween-40, and Tween-80. In some embodiments, the Triton is Triton X-100 or Triton X-1 14. In some embodiments, the IGEPAL is selected from the group consisting of IGEPAL CO-520, IGEPAL CO-630, and IGEPAL CO-720.

In some embodiments, the detergent or detergents reduce the structural integrity of the eukaryotic cell.

In some embodiments, the eukaryotic cell lysis composition (or mixture) comprises a salt. In some embodiments, the salt is a divalent salt. In some embodiments, the salt is an alkali earth metal salt, such as a magnesium salt, a calcium salt, a strontium salt, or a barium salt. In some embodiments, the salt comprises a magnesium salt. In accordance with some embodiments, the magnesium salt is selected from the group consisting of MgCl₂, MgCO₃, MgSO₄, and MgBr₂.

In some embodiments, a concentration of the salt (e.g., a magnesium salt) in the composition or mixture is greater than or equal to 0.1 mM, greater than or equal to 1 mM, greater than or equal to 5 mM, greater than or equal to 10 mM, greater than or equal to 15 mM, greater than or equal to 20 mM, greater than or equal to 25 mM, greater than or equal to 30 mM, greater than or equal to 35 mM, or greater than or equal to 70 mM. According to some embodiments, a total concentration of the salt (e.g., a magnesium salt) in the composition or mixture is less than or equal to 500 mM, less than or equal to 300 mM, less than or equal to 100 mM, less than or equal to 75 mM, less than or equal to 50 mM, less than or equal to 45 mM, less than or equal to 40 mM, less than or equal to 35 mM, less than or equal to 30 mm, less than or equal to 25 mM, less than or equal to 20 mM, or less than or equal to 15 mM. Combinations of the above-referenced ranges are also possible (e.g., a total concentration of the salt (e.g., a magnesium salt) between 1 mM and 50 mM, inclusive, or between 5 mM and 25 mM, inclusive, are possible). Other ranges are also possible.

In some embodiments, at least one anti-foaming agent is combined with the eukaryotic cell lysis solution. Anti-foaming agents include, but are not limited to, Antifoam A, Antifoam 204, Antifoam B, Antifoam C, Antifoam Y-30, Antifoam SE-15, and simethicone-based antifoams.

In some embodiments, the mixture contains less than about 0.15 M of monovalent salts. Without wishing to be bound by theory, in some embodiments, when the mixture contains less than about 0.15 M of monovalent salts there is an induction of osmotic stress. In some embodiments, the volume ratio of the eukaryotic cell lysis solution to the sample is about 0.25:1, 0.5:1, 0.75:1, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, or any ratio between any two of these ratios.

In some embodiments, the eukaryotic cell lysis reaction is carried out at about room temperature. In some embodiments, the eukaryotic cell lysis reaction is carried out at between about 5° C. to 20° C., about 9° C. to 16° C., or about 12° C. to 13° C. In some embodiments, the eukaryotic cell lysis reaction is carried at temperatures between about 25° C. to 75° C., about to 70° C., about 35° C. to 65° C., about 40° C. to 60° C., or about 45° C. to 55° C.

In some embodiments, the eukaryotic cell lysis reaction is carried out for between about 0.01-20 minutes, between about 0.1-9.0 minutes, between about 1.0-8.0 minutes, between about 2.0-7.0 minutes, between about 3.0-6.0 minutes, between about 4.0-5.0 minutes. In some embodiments, the eukaryotic cell lysis process is stopped after about 5 minutes.

In some embodiments, the eukaryotic cell lysis solution does not contain a buffering agent. In other embodiments, the eukaryotic cell lysis solution contains a buffering agent. Examples of buffering agents include, but are not limited to 2-(N-morpholino)ethanesulfonic acid (MES), 2-Bis(2-hydroxyethyl)amino-2-(hydroxymethyl)-1,3-propanediol (Bis-Tris), 3-(-morpholino)propanesulfonic acid (MOPS), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), tris(hydroxymethyl)aminomethane) (TRIS), Arginine, Lysine, Sodium Phosphate, Potassium Phosphate, Sodium Acetate, Sodium Carbonate/Bicaronate buffers, Sodium Acetate, N-cyclohexyl-2-hydroxyl-3-aminopropanesulfonic acid (CAPSO), N-(2-Hydroxyethyl)piperazine-N′-(4-butanesulfonic acid) (HEPBS), N-methylpiperazine, piperazine, diethanolamine, and propane 1,3-diamino.

In some embodiments, the pH of the eukaryotic cell lysis reaction is between about a pH of 6 to 9.5. In some embodiments, the pH is at or near neutral. Selective lysis of eukaryotic cells at a pH between about 6 to 9.5 or near neutral is in contrast to current methods, which emphasize alkaline conditions for eukaryotic cell lysis reactions (e.g., at pH 9.5-14). In some embodiments, performing the eukaryotic cell lysis reaction at a pH between about 6 to 9.5 or near neutral is advantageous over current methods known in the art due to an increase in the viability and/or structural integrity of microbial cells in the presence of some surfactants.

In some embodiments, the methods for eukaryotic cell lysis reactions described herein are advantageous over current methods known in the art because the eukaryotic cell lysis reaction methods described herein are suitable for automation in an integrated device. In some embodiments, the eukaryotic cell lysis reaction is terminated by adding a lysis termination solution that increases the electrolyte strength, and if necessary, the pH of the reaction, back to roughly physicological conditions.

Selective Lysis of Eukaryotic Cells while Leaving Borrelia Intact

In some embodiments, when the suspected pathogen is one or more species of Borrelia, specialized lysis solutions and methods are used. Without wishing to be bound by theory, the selective methods described herein may provide for (i) a selective destabilization of the eukaryote cell membrane without destabilizing the cell membrane of Borrelia cells; and (ii) inducing lysis of destabilized eukaryotic cells via osmotic stress. Indeed, cell permeability to certain ions and other molecules is dependent on the organization of membrane lipids and proteins, and destabilization of a cell's membrane alters the organization of the cell membrane's lipids and proteins, thus altering its permeability. It has surprisingly been found that the compositions described herein may be capable of destabilizing a eukaryotic cell, e.g., white blood cell (WBC) membrane while not achieving the same to a cell of interest, for example Borrelia. Once the eukaryotic cell membrane has been destabilized, cell rupturing is induced by altering (i.e., lowering) the electrolyte strength of the solution and/or adjusting pH. This can be done in one or multiple steps. Thus, destabilization and rupturing of eukaryotic cells releases their genomic material while Borrelia cells remain intact.

In some embodiments, the methods described herein comprise contacting the sample with an ultrasensitive eukaryotic cell lysis solution or composition described herein.

In some embodiments, the lysis solution or composition comprises one or more chemical lysis agents. In some embodiments, the chemical lysis agents may include, but are not limited to, detergents such as cationic detergents, non-ionic detergents, and zwitterionic detergents. In some embodiments, the chemical lysis agent comprises a lipid. In some embodiments, the chemical lysis agent comprises a fos-choline.

In some embodiments, the eukaryotic lysis solution or composition comprises a chemical lysis agent comprising a compound of Formula I:

wherein R₁ is selected from the group consisting of optionally substituted, branched or unbranched, saturated or unsaturated C₁-C₈ aliphatic; optionally substituted, saturated or unsaturated C₃-C₁₄ carbocyclic; optionally substituted, saturated or unsaturated 3-8 membered heterocyclic; optionally substituted, branched or unbranched, saturated or unsaturated ((R_(a))_(q)—(C═O)—(R_(a))_(q))_(p); optionally substituted C₆-C₁₄ aryl; and optionally substituted 3-8 membered heteroaryl; and/or any suitable combinations thereof;

wherein R₂ is selected from the group consisting of hydrogen; optionally substituted, branched or unbranched, saturated or unsaturated C₁-C₂₈ aliphatic; optionally substituted, branched or unbranched, saturated or unsaturated —(R_(b)—(O—R_(b))_(n)—O—R_(b))_(p); optionally substituted, branched or unbranched, saturated or unsaturated —(R_(b)—(O—R_(b))_(n)—NH—R_(b))_(p); optionally substituted, branched or unbranched, saturated or unsaturated —(R_(b)—(O—R_(b)—O)_(n)—S—R_(b))_(p); optionally substituted, branched or unbranched, saturated or unsaturated —(R_(b)—(S—R_(b))_(n)—S—R_(b))_(p); optionally substituted C₆-C₁₄ aryl; optionally substituted 3-8 membered heteroaryl; optionally substituted, saturated or unsaturated C₃-C₁₄ carbocyclic; optionally substituted, saturated or unsaturated 3-8 membered heterocyclic; optionally substituted, branched or unbranched, saturated or unsaturated —(C═O)—(R_(b)); optionally substituted, branched or unbranched, saturated or unsaturated —((R_(a))_(q)—O—(R_(a))_(q))_(p)—; optionally substituted, branched or unbranched, saturated or unsaturated —((R_(a))_(q)—NH—(R_(a))_(q))_(p)—; optionally substituted, branched or unbranched, saturated or unsaturated —((R_(a))_(q)—N(R_(a))—(R_(a))_(q))_(p)—; and optionally substituted, branched or unbranched, saturated or unsaturated —((R_(a))_(q)—S—(R_(a))_(q))_(p)—; and/or any suitable combinations thereof;

wherein each occurrence of R a is independently C₁-C₈ aliphatic or C₆-C₁₄ aryl;

wherein each occurrence of R_(b) is independently C₁-C₁₅ aliphatic or C₆-C₁₄ aryl;

wherein each occurrence of subscript q is independently an integer between 0 and 1,

wherein each occurrence of subscript p is independently an integer between 1 and 6, inclusive; and

wherein each occurrence of subscript n is independently an integer between 0 and 14, inclusive.

In some embodiments, R₁ is independently selected from the group consisting of optionally substituted, branched or unbranched C₁-C₈ alkyl; optionally substituted, branched or unbranched C₂-C₈ alkenyl; and optionally substituted, branched or unbranched C₂-C₈ alkynyl.

In accordance with some embodiments, R₁ is optionally substituted, branched or unbranched C₁-C₈ alkyl.

According to some embodiments, R₁ is C₂ alkyl.

In accordance with some embodiments, R₂ is independently selected from the group consisting of optionally substituted, branched or unbranched C₁-C₂₈ alkyl, optionally substituted, branched or unbranched C₂-C₂₈ alkenyl, optionally substituted, branched or unbranched C₂-C₂₄ alkynyl, optionally substituted C₆-C₁₄ aryl, optionally substituted C₃-C₁₄ cycloalkyl, optionally substituted —CH₂—(OCH₂—CH₂)₁₁₀—CH₃, optionally substituted —CH₂—(OCH₂—CH₂)_(n)NHCH₃, optionally substituted —CH₂—(OCH₂—CH₂O)_(n)SCH₃, optionally substituted —CH₂—(SCH₂—CH₂)_(n)SCH₃, and optionally substituted —OC—(CH₂)_(n)CH₃.

In some embodiments, R₂ is independently selected from the group consisting of optionally substituted, branched or unbranched C₁-C₂₈ alkyl and optionally substituted, branched or unbranched C₂-C₂₈ alkenyl.

According to some embodiments, R₂ is independently selected from the group consisting of optionally substituted, branched or unbranched C₄-C₁₆ alkyl and C₁₁ alkenyl.

In some embodiments, R₂ is C₁₆ alkyl.

In accordance with some embodiments, the compound of Formula 1 is selected from the group consisting of:

Fos-Choline-8,

Fos-Choline-9,

Fos-Choline-10,

Fos-Choline-11,

Fos-Choline-12,

Fos-Choline-13,

Fos-Choline-14,

Fos-Choline-15,

Fos-Choline-16,

Fos-Choline-Unsat-11-10,

Fos-Choline-ISO—9, and

Fos-Choline-ISO—11.

In some embodiments, the compound of Formula 1 is

Fos-Choline-16.

In some embodiments, a composition is provided. The composition comprises a eukaryotic cell chemical lysis agent (e.g., a compound of Formula I) and one or more optional components as described herein. According to some embodiments, a concentration of the chemical lysis agent (such as a compound of Formula I) in the composition is greater than or 1 mM, greater than or equal to 5 mM, greater than or equal to 10 mM, greater than or equal to 25 mM, greater than or equal to 50 mM, greater than or equal to 100 mM, greater than or equal to 200 mM, greater than or equal to 300 mM, greater than or equal to 400 mM, greater than or equal to 500 mM, or greater than or equal to 1,000 mM. In some embodiments, a concentration of the chemical lysis agent (such as a compound of Formula I) in the composition is less than or equal to 1,000 mM, less than or equal to 500 mM, less than or equal to 250 mM, less than or equal to 200 mM, less than or equal to 150 mM, less than or equal to 100 mM, less than or equal to 50 mM, less than or equal to 25 mM, less than or equal to 10 mM, less than or equal to 5 mM, or less than or equal to 1 mM. Combinations of the above-referenced ranges are also possible (e.g., a concentration of the chemical lysis agent (such as a compound of Formula 1) between 1 mM and 250 mM, inclusive, a concentration of between 1 mM and 25 mM, inclusive, or a concentration of between 10 mM and 250 mM, inclusive, are possible). Other ranges are also possible.

In accordance with some embodiments, if R₂ in a compound of Formula I contains 10 or fewer non-hydrogen atoms (e.g., C, O, N, and/or S), a concentration of the compound of Formula I in the composition is greater than or equal to 25 mM, greater than or equal to 50 mM, greater than or equal to 100 mM, or greater than or equal to 200 mM, or greater than or equal to 1,000 mM. In some embodiments, if R₂ in a compound of Formula I contains 10 or fewer non-hydrogen atoms (e.g., C, O, N, and/or S), a concentration of the chemical lysis agent (such as a compound of Formula I) in the composition is less than or equal 1,000 mM, is less than or equal to 250 mM, less than or equal to 200 mM, less than or equal to 150 mM, less than or equal to 100 mM, less than or equal to 50 mM, or less than or equal to 25 mM. Combinations of the above-referenced ranges are also possible (e.g., a concentration of the chemical lysis agent (such as a compound of Formula 1) between 25 mM and 250 mM, inclusive, is possible). Other ranges are also possible.

In other embodiments, if R₂ in a compound of Formula I contains more than 10 non-hydrogen atoms (e.g., C, O, N, and/or S), a concentration of the compound of Formula I in the composition is greater than or equal to 1 mM, greater than or equal to 5 mM, or greater than or equal to 10 mM, or greater than or equal to 50 mM, or greater than 50 mM or equal to 100 mM. In some embodiments, if R₂ in a compound of Formula I contains more than 10 non-hydrogen atoms (e.g., C, O, N, and/or S), a concentration of the chemical lysis agent in the composition (such as a compound of Formula I) is less than or equal to 100 mM, is less than or equal to 50 mM, is less than or equal to 25 mM, less than or equal to 10 mM, less than or equal to 5 mM. Combinations of the above-referenced ranges are also possible (e.g., a concentration of the chemical lysis agent (such as a compound of Formula 1) between 1 mM and 25 mM, inclusive, is possible). Other ranges are also possible.

In some embodiments, the total concentration of the chemical lysis agent (such as a compound of Formula I) in the ultrasensitive eukaryotic cell lysis reaction (e.g., ultrasensitive eukaryotic cell lysis solution combined with the sample (hereinafter, the “mixture”)) is greater than or equal to greater than or equal to 0.25 mM, greater than or equal to 1 mM, greater than or equal to 5 mM, greater than or equal to 10 mM, greater than or equal to 25 mM, greater than or equal to 50 mM, greater than or equal to 100 mM, or greater than or equal to 200 mM. In some embodiments, a total concentration of the chemical lysis agent (such as a compound of Formula I) in the mixture is less than or equal to 250 mM, less than or equal to 200 mM, less than or equal to 150 mM, less than or equal to 100 mM, less than or equal to 50 mM, less than or equal to 25 mM, less than or equal to 10 mM, less than or equal to 5 mM, or less than or equal to 1 mM. Combinations of the above-referenced ranges are also possible (e.g., a total concentration of the chemical lysis agent in the mixture (such as a compound of Formula 1) between 0.25 mM and 250 mM, inclusive, a total concentration of between 1 mM and 25 mM, inclusive, or a total concentration of between 10 mM and 250 mM, inclusive, are possible). Other ranges are also possible.

In accordance with some embodiments, if R₂ in a compound of Formula I contains 10 or fewer non-hydrogen atoms (e.g., C, O, N, and/or S), a total concentration of the compound of Formula I in the mixture is greater than or equal to 20 mM, greater than or equal to 50 mM, greater than or equal to 100 mM, or greater than or equal to 200 mM. In some embodiments, if R₂ in a compound of Formula I contains 10 or fewer non-hydrogen atoms (e.g., C, O, N, and/or S), a total concentration of the chemical lysis agent (such as a compound of Formula I) in the mixture is less than or equal to 250 mM, less than or equal to 200 mM, less than or equal to 150 mM, less than or equal to 100 mM, less than or equal to 50 mM. Combinations of the above-referenced ranges are also possible (e.g., a total concentration of the chemical lysis agent (such as a compound of Formula 1) between 20 mM and 250 mM, inclusive, is possible). Other ranges are also possible.

In other embodiments, if R₂ in a compound of Formula I contains more than 10 non-hydrogen atoms (e.g., C, O, N, and/or S), a total concentration of the compound of Formula I in the mixture is greater than or equal to 0.25 mM, greater than or equal to 1 mM, greater than or equal to 5 mM, greater than or equal to 10 mM, greater than or equal to 25 mM, or greater than or equal to 50 mM. In some embodiments, if R₂ in a compound of Formula I contains more than 10 non-hydrogen atoms (e.g., C, O, N, and/or S), a total concentration of the chemical lysis agent in the mixture (such as a compound of Formula I) is less than or equal to 50 mM, less than or equal to 25 mM, less than or equal to 10 mM, or less than or equal to 5 mM. Combinations of the above-referenced ranges are also possible (e.g., a total concentration of the chemical lysis agent (such as a compound of Formula 1) between 0.25 mM and 25 mM, inclusive, is possible). Other ranges are also possible.

In some embodiments, the eukaryotic chemical lysis agent (either as a group or individually, or any combination thereof) are stored in dry or pelleted form, where upon re-suspension of the respective eukaryotic chemical lysis agent, the agent reaches the concentrations identified above.

According to some embodiments, the eukaryotic cell lysis mixture and/or composition comprises a pH greater than or equal to 6, greater than or equal to 7, greater than or equal to 8, greater than or equal to 9, or greater than or equal to 10. In accordance with some embodiments, the eukaryotic cell lysis mixture or composition comprises a pH of less than or equal to 14, less than or equal to 13, less than or equal to 12, less than or equal to 11, less than or equal to 10, or less than or equal to 9. Combinations of the above-referenced ranges are also possible (e.g., a pH between 8 and 11, inclusive). Other ranges are also possible.

In some embodiments, the eukaryotic cell lysis reaction is performed at a pH of greater than or equal to 6, greater than or equal to 7, greater than or equal to 8, greater than or equal to 9, or greater than or equal to 10. In accordance with some embodiments, the eukaryotic cell lysis reaction is performed at a pH of less than or equal to 14, less than or equal to 13, less than or equal to 12, less than or equal to 11, less than or equal to 10, or less than or equal to 9. Combinations of the above-referenced ranges are also possible (e.g., a pH between 8 and 11, inclusive). Other ranges are also possible.

In some embodiments, the eukaryotic cell lysis composition or mixture also includes one or more of the following: detergents, salts, buffering agents, water, and metal chelators.

In some embodiments, multiple eukaryotic cell lysis solutions are used. In some embodiments, the multiple eukaryotic cell lysis solutions are added in a step wise fashion. In some embodiments, only a single eukaryotic cell lysis solution is used.

In some embodiments, the eukaryotic cell lysis reaction is heated to between about to 50° C., about 20° C. to 45° C., about 25° C. to 40° C., or about 30° C. to 35° C. In some embodiments, the eukaryotic cell lysis reaction is performed at room temperature. According to some embodiments, the eukaryotic cell lysis composition (or mixture) comprises a salt. In some embodiments, the salt is a divalent salt. In some embodiments, the salt is an alkali earth metal salt, such as a magnesium salt, a calcium salt, a strontium salt, or a barium salt. In some embodiments, the salt comprises a magnesium salt. In accordance with some embodiments, the magnesium salt is selected from the group consisting of MgCl₂, MgCO₃, MgSO₄, and MgBr₂.

In some embodiments, a concentration of the salt (e.g., a magnesium salt) in the composition or mixture is greater than or equal to 0.1 mM, greater than or equal to 1 mM, greater than or equal to 5 mM, greater than or equal to 10 mM, greater than or equal to 15 mM, greater than or equal to 20 mM, greater than or equal to 25 mM, greater than or equal to mM, greater than or equal to 35 mM, or greater than or equal to 70 mM. According to some embodiments, a total concentration of the salt (e.g., a magnesium salt) in the composition or mixture is less than or equal to 500 mM, less than or equal to 300 mM, less than or equal to 100 mM, less than or equal to 75 mM, less than or equal to 50 mM, less than or equal to 45 mM, less than or equal to 40 mM, less than or equal to 35 mM, less than or equal to 30 mm, less than or equal to 25 mM, less than or equal to 20 mM, or less than or equal to 15 mM. Combinations of the above-referenced ranges are also possible (e.g., a total concentration of the salt (e.g., a magnesium salt) between 1 mM and 50 mM, inclusive, or between 5 mM and 25 mM, inclusive, are possible). Other ranges are also possible.

In some embodiments, the one or more salts is stored in dry or pelleted form, where upon re-suspension of the respective salt, the salt reaches the concentrations identified above.

According to some embodiments, a mixture described herein is a blood-based mixture comprising the lysis solution or composition and blood.

In some embodiments, the blood-based mixture comprises a blood-to-lysis solution volumetric ratio of 1 to greater than or equal to 0.5, greater than or equal to 0.75, greater than or equal to 1, greater than or equal to 1.25, greater than or equal to 1.5, greater than or equal to 1.75, greater than or equal to 2, greater than or equal to 2.25, greater than or equal to 2.5, greater than or equal to 2.75, greater than or equal to 3, or greater than or equal to 3.25. In accordance with some embodiments, the blood-based mixture comprises a blood-to-lysis solution volumetric ratio of 1 to less than or equal to 3.75, less than or equal to 3.5, less than or equal to 3.25, less than or equal to 3, less than or equal to 2.75, less than or equal to 2.5, less than or equal to 2.25, less than or equal to 2, less than or equal to 1.75, less than or equal to 1.5, less than or equal to 1.25, or less than or equal to 1. Combinations of the above-referenced ranges are also possible (e.g., a blood-to-lysis solution volumetric ratio between 1:0.75 and 1:3.5, inclusive). Other ranges are also possible.

According to some embodiments, the blood-based mixture comprises greater than or equal to 15%, greater than or equal to 20%, greater than or equal to 25%, greater than or equal to 30%, greater than or equal to 35%, greater than or equal to 40%, greater than or equal to 50%, or greater than or equal to 55% of the blood by volume. In some embodiments, the blood-based mixture comprises less than or equal to 65%, less than or equal to 60%, less than or equal to 55%, less than or equal to 50%, less than or equal to 45%, less than or equal to 40%, less than or equal to 35%, less than or equal to 30%, or less than or equal to 25% of the blood by volume. Combinations of the above-referenced ranges are also possible (e.g., between 20% and 60%, inclusive, of the blood by volume).

In some embodiments, the eukaryotic cell lysis solution or composition does not contain a buffering agent. In other embodiments, the eukaryotic cell lysis solution or composition comprises a buffering agent. Examples of buffering agents include, but are not limited to 2-(N-morpholino)ethanesulfonic acid (MES), 2-Bis(2-hydroxyethyl)amino-2-(hydroxymethyl)-1,3-propanediol (Bis-Tris), 3-(-morpholino)propanesulfonic acid (MOPS), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), tris(hydroxymethyl)aminomethane) (TRIS), Sodium Phosphate, Potassium Phosphate, Sodium Acetate, Sodium Carbonate/Bicarbonate buffers, Sodium Acetate, N-cyclohexyl-2-hydroxyl-3-aminopropanesulfonic acid (CAPSO), N-(2-Hydroxyethyl)piperazine-N′-(4-butanesulfonic acid) (HEPBS), N-methylpiperazine, piperazine, diethanolamine, and propane 1,3-diamino.

In some embodiments, the eukaryotic cell lysis solution or composition comprises an amino acid. In some embodiments, the amino acid comprises alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and/or valine. In some embodiments, a concentration of the amino acid in the composition or the mixture is greater than or equal to 0.01 M, greater than or equal to 0.1M, greater than or equal to 0.2M, greater than or equal to 0.5M, greater than or equal to 1M, greater than or equal to or greater than or equal to 10M. In some embodiments, a concentration of the amino acid in the composition or the mixture is less than or equal to 0.01 M, less than or equal to 0.1M, less than or equal to 0.2M, less than or equal to 0.5M, less than or equal to 1M, less than or equal to 5M, or less than or equal to 10M. Combinations of the above-referenced ranges are also possible. In some embodiments, a concentration of the amino acid in the composition or the mixture is between about 0.01M and 0.2M, between about 0.1M-1M, between about 0.5M-5M, or between about 1M-10M. Other ranges are also possible.

Removing Eukaryotic DNA/RNA

In some embodiments, the eukaryotic DNA released by the lysis of the eukaryotic cells is removed from the sample by centrifugation. In some embodiments, the sample is centrifuged and the supernatant containing the eukaryotic DNA is removed from the pellet containing the intact microbial cells.

As is known to those skilled in the art, an efficient and effective manner of concentrating microbial cells is centrifugation. Post-centrifugation of microbial cells, a pellet is formed which allows a user to conduct a multitude of processes inclusive of removal of the supernatant (i.e. buffer exchange). In some embodiments, the sample is centrifuged at a speed of 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2200, 2400, 2500, 2600, 2800, 3000, 3500, 4000, or 5000 g, e.g., 2000 g. In some embodiments, the sample is centrifuged for 1-30 minutes, e.g, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 19, 20 minutes, e.g., 10 minutes.

Without wishing to be bound by theory, in cases where the microbial load is low, while a microbial pellet is produced, it may be unstable due an inability to reach a critical mass. This pellet may be disrupted thereby reducing sensitivity or resulting in a failed assay.

Thus, in some embodiments, one or more particles are added to the sample prior to centrifugation. Inert microparticles are used to produce, in conjunction with the microorganisms in the sample, a more stable pellet will withstand (i.e. remain intact during) post-centrifugation procedures described herein. This is demonstrated in FIG. 3 . As is shown in FIG. 3 , without the addition of particles, initially the microbial cells are randomly dispersed within the medium. After centrifugation a small pellet is formed at the bottom of the cone consisting of all the microbial cells. The pellet is readily disturbed, which in an assay would either (1) result in a possible false-negative (i.e. missed infection), or (2) result in a lower signal. However, as is shown in FIG. 3 , when particles are added to the sample, this result in a larger, naturally more stable pellet post-centrifugation. The exemplary sample in FIG. 3 contains three particle types incorporated (all inert, polystyrene) of the following sizes: (1) ‘Large’—being of a typical diameter in the 5-8 μm range; (2) ‘Mid-size’—being of a typical diameter in the 1 μm range; and (3) ‘Small’— being of a typical diameter in the range.

In some embodiments, the particles are microparticles. In some embodiments, the microparticles have a diameter of 0.01-100 μm, e.g., 0.05-20 μm or 0.1-10 μm. In some embodiments, the microparticles have a diameter of at least 0.01, 0.05, 0.1, 0.2, 0.5, 1, 2, 3, 4, 6, 7, 8, 9, 10, 20, 50 or 100 μm. In some embodiments, the microparticles have a diameter of less than or equal to 100, 50, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.2, 0.1, 0.05, or 0.01 μm. In some embodiments, microparticles of more than one diameter are added to the sample, e.g., microparticles of 2, 3, 4, or 5, or more different diameters are added to the sample. In some embodiments, microparticles are added to the sample having one or more of the following diameters: (i) 4-10 μm, e.g., 5-8 μm; (ii) 0.5-2 μm, e.g., about 1 μm; and (iii) 0.05-1 μm, e.g., 0.2 μm.

In some embodiments, the particles are a polymer. In some embodiments, the particles are polystyrene, silica, silica dioxide, latex, iron, or a melamine resin. In some embodiments, the particles are magnetic.

Given the need to reach ultra-sensitive detection capabilities, the loss of even a single microbial cell should be avoided. A specific concern is one where, due to poorly implemented practices, the pellet is disturbed. In such a situation one of two event may occur: (i) a complete loss of target acquisition capability, resulting in a false-negative; or (ii) a partial loss of target acquisition capability, resulting in a reduced signal. To address this, in some embodiments, a control is added to the sample prior to centrifugation. A specific type of an ‘Internal Control’ (or IC) is designed into this system where: (1) the absence of an IC signal, regardless of the target signal, would render a null result, and (2) the presence of an IC signal would indicate a successful test, regardless of the target signal.

In some embodiments, the IC is a live microorganism having one or more of the following characteristics:

(i) The IC is a lyophilized pellet containing a known and repeatable load of the IC, which will generate a positive signal when the microbial pellet is not disturbed;

(ii) The IC lyophilized pellet is stored in the specimen collection tube such that upon introduction of the sample, the lyophilized pellet is reconstituted and mixed with the sample;

(iii) The IC is comprised of a single microorganism which is not known to be a common human pathogen and should not be found in the sample;

(iv) The IC is comprised of a single microorganism containing a unique gene or plasmid that is readily known and is capable of being PCR amplified in a highly specific manner; and

(v) The IC is comprised of a single microorganism that is readily lysed in the microbial lysis step described below.

In some embodiments, the eukaryotic DNA released by the lysis of the eukaryotic cells is removed from the sample by size exclusion chromatography.

In some embodiments, the separation of the eukaryotic genomic material from the intact microbial cells in the mixture, is performed through “selective capture” of eukaryotic genomic material or immobilization of the eukaryotic DNA without or only minimally capturing or immobilization of the intact microbial cells, eukaryotic cellular debris, or other non-nucleic acid material. In some embodiments, the eukaryotic genomic material captured is eukaryotic DNA and/or RNA.

In some embodiments, an anion exchange resin is used to capture/immobilize eukaryotic genomic material. In some embodiments, an anion exchange resin is one or more weak anion-exchange resins (WAX). Examples of WAX include, but are not limited to, carboxymethyl (CM), diethylaminopropyl (ANX), diethylethanolamine (DEAE), Amberlite Ira67, Purolite A847, Amberlite Ira96, Amberlite IRA96SB, Dowex Marathon WBA, Dowex Upcore Mono WB-500, Purolite A835, Dowex Monosphere 77, and Dowex Monosphere 66. In some embodiments, the WAX resin contains at least one tertiary amine functional group.

In some embodiments, the WAX resin contains at least one secondary amine functional group. In some embodiments, the WAX resin contains at least one secondary amine and at least one tertiary functional group.

In some embodiments, an anion exchange resin is one or more strong anion-exchange resins (SAX). Examples of SAX include, but are not limited to, -0-CH₂—CHOH—CH₂-0-CH₂—CHOH—CH₂—N⁺(CH₃)₃, Amberjet Up4000, Amberjet 9000 OH, Amberlite FPA40 CI, and Dowex Upcore Mono MA-600. In some embodiments a SAX based resin contains a quaternary amine functional group.

In some embodiments, the anion exchange resin is a combination of at least one WAX and at least one SAX.

In some embodiments, the form of the anion exchange resin is selected from fibers, membranes, sorbents, gels, polymers, and filters. In some embodiments, the sample with the lysed eukaryotic cells is passed through or contacted with the anion exchange resin. In some embodiments, the anion exchange resin is in a solution.

In some embodiments, the anion exchange resin is conjugated to a support substrate. Examples of a support substrate include, but are not limited to, a particle, a bead, a surface, or a sphere. In some embodiments, the support substrate is magnetic, e.g., a magnetic particle or bead. In some embodiments, the anion exchange resin is conjugated to an support substrate is in a solution.

In some embodiments, the support substrate comprises silica, glass, metal, iron, latex, polystyrene-based material, cellulose-based material, agarose-based material, dextran-based material, methacrylate-based material, sepharose-based material, or a combination thereof. In some embodiments, the support substrate is porous.

In some embodiments, the support substrate is a bead or sphere has a diameter between about 10 to 100 μm, between about 20 to 90 μm, between about 30 to 80 μm, between about 40 to 70 μm, or between about 50 to 60 μm.

In another embodiment, the support substrate is a bead or sphere have a diameter between about 0.01 to 10 μm, about 0.1 to 9.0 μm, about 1.0 to 8.0 μm, about 2.0 to 7.0 μm, about 3.0 to 6.0 μm, or between about 4.0 to 5.0 μm.

In some embodiments, the anion exchange resin is WAX and the support substrate is a magnetic microparticle having a diameter of 0.1-5 μm, e.g., about 1 μm. In some embodiments, the mixture is incubated with the anion exchange resin between about 0.1 to 10 minutes, between about 2 to 9 minute, between about 3 to 8 minutes, between about 4 to 7 minutes, or between about 5 to 6 minutes. In some embodiments, the mixture is incubated with the anion exchange resin between about 10 to 30 minutes, between about 12 to 28 minutes, between about 15 to 25 minutes, between about 18 to 23 minutes, or between about 19 to 22 minutes. In some embodiments, the mixture is incubated with the anion exchange resin for less than 1 minute.

In some embodiments, the anion exchange resin is permanently immobilized on the support substrate. In some embodiments, the immobilized anion exchange resin is contacted and/or incubated with the mixture and then the mixture is removed.

In some embodiments, at least one anion exchange resin conjugated to a support substrate, e.g., a bead or a particle, is contacted and/or incubated with the mixture. In some embodiments, after contacting and/or incubation with the mixture, the anion exchange resin conjugated to a support substrate is removed from the mixture. In another embodiment, after contacting and/or incubation with the mixture, the anion exchange resin conjugated to a support substrate is immobilized and the mixture is removed. By way of example, but not by way of limitation, in some embodiments, the anion exchange resin conjugated to a support substrate is selectively immobilized when the support substrate is a magnetized or metal particle and the magnetized or metal particle is exposed to a magnet or magnetic field.

In some embodiments, contacting and/or incubating the mixture with the anion exchange resin extracts eukaryotic DNA, e.g., human DNA (hDNA), and/or RNA from the mixture. In some embodiments, the eukaryotic DNA (and/or RNA) binds to the anion exchange resin. In some embodiments, the anion exchange resin extracts between about 5% to 100%, between about 10% to 99%, between about 15% to 85%, between about 20% to 80%, between about 25% to 75%, between about 30% to 70%, between about 35% to 65%, between about 40% to 60%, or between about 45% to 55% of the eukaryotic DNA (and/or RNA), e.g., hDNA, from the mixture. In some embodiments, the anion exchange resin extracts over 95% of the eukaryotic DNA from the mixture.

Lysing of Microorganisms

In some embodiments, wherein it is desirable to assay the microorganisms listed in Tables 1-33 inclusive for Borrelia and/or additional bacteria and/or fungi, it is preferred to ensure that the microbial lysis step be effective on all targets. A similar process to the one disclosed here, is illustrated in detail in WO 2016/044621A1. In some embodiments, the mixture with the eukaryotic DNA removed (hereinafter “isolated microbial cell sample”) contains one or more microbial cells. In some embodiments, the isolated microbial cell sample is subjected to further processing. In some embodiments, the isolated microbial cell sample is contacted with a microbial cell lysis solution.

In some embodiments, the microbial cells are lysed using a lysis solution including one or more chemical lysis agents. In some embodiments, the chemical lysis agents include, but are not limited to, cationic detergents, non-ionic detergents, zwitterionic detergents, and enzymes.

In some embodiments, the microbial lysis reaction is performed at a pH between about 6 to 9 or at a neutral pH.

In some embodiments, the microbial lysis solution also includes one or more of the following: enzymes, detergents, and other components such as salts, buffering agents, and metal chelators.

In some embodiments, multiple lysis solutions are used. In some embodiments, the multiple lysis buffers are added in a step wise fashion. In some embodiments, only a single microbial lysis solution is used.

In some embodiments, the microbial lysis reaction is heated to between about 15° C. to about 20° C. to 45° C., about 25° C. to 40° C., or about 30° C. to 35° C. In some embodiments, the microbial lysis reaction is performed at room temperature.

In some embodiments, the microbial lysis solution includes one or more of the following enzymes or enyzme groups: lysozyme, lyticase, zymolyase, mutanolysin, and lysostaphin. In some embodiments, the one or more enzymes are stored in dry or pelleted form, where upon re-suspension of the respective enzyme, the enzyme reaches the concentrations identified below.

In some embodiments, the lysozyme concentration in the microbial lysis solution is between about 5 to 200 mg/ml, about 1 to 150 mg/ml, 5 to 175 mg/ml, about 15 to 140 mg/ml, about 20 to 100 mg/ml, about 30 to 95 mg/ml, about 45 to 75 mg/ml, about 50 to 62 mg/ml, or between any two of the previously disclosed concentrations.

In some embodiments, the lysozyme concentration in the microbial lysis reaction (e.g., a solution including the microbial lysis solution and the isolated microbial cell sample) is between about 0.01 to 1 mg/ml, about 0.1 to 10 mg/ml, 0.5 to 15 mg/ml, about 1 to 20 mg/ml, about 0.3 to 8 mg/ml, about 0.7 to 7 mg/ml, about 0.2 to 0.9 mg/ml, about 0.05 to mg/ml, or between any two of the previously disclosed concentrations.

In some embodiments, the lyticase concentration in the microbial lysis solution is between about 500 to 50,000 U/ml, about 250 to 10,000 U/ml, 425 to 8,000 U/ml, about 300 to 6,000 U/ml, about 400 to 5,000 U/ml, about 1,000 to 4,750 U/ml, about 1,500 to 4,500 U/ml, about 2,000 to 6,500 U/ml, about 2,500 to 5,500 U/ml, about 3,000 to 15,000 U/ml, or between any two of the previously disclosed concentrations.

In some embodiments, the lyticase concentration in the microbial lysis reaction is between about 1 to 1000 U/ml, about 5 to 200 U/ml, 20 U to 800 U/ml, about 30 to 700 U/ml, about 40 to 600 U/ml, about 50 to 500 U/ml, about 60 to 400 U/ml, about 70 to 300 U/ml, about 80 to 200 U/ml, about 90 to 100 U/ml, or between any two of the previously disclosed concentrations.

In some embodiments, the zymolyase concentration in the microbial lysis solution is between about 500 to 50,000 U/ml, about 250 to 10,000 U/ml, 425 U to 8,000 U/ml, about 300 to 6,000 U/ml, about 400 to 5,000 U/ml, about 1,000 to 4,750 U/ml, about 1,500 to 4,500 U/ml, about 2,000 to 6,500 U/ml, about 2,500 to 5,500 U/ml, about 3,000 to 15,000 U/ml, or between any two of the previously disclosed concentrations.

In some embodiments, the zymolyase concentration in the microbial lysis reaction is between about 1 to 1000 U/ml, about 5 to 200 U/ml, 20 U to 800 U/ml, about 30 to 700 U/ml, about 40 to 600 U/ml, about 50 to 500 U/ml, about 60 to 400 U/ml, about 70 to 300 U/ml, about 80 to 200 U/ml, about 90 to 100 U/ml, or between any two of the previously disclosed concentrations.

In some embodiments, the mutanolysin concentration in the microbial lysis solution is between about 500 to 50,000 U/ml, about 250 to 10,000 U/ml, 425 to 8,000 U/ml, about 300 to 6,000 U/ml, about 400 to 5,000 U/ml, about 1,000 to 4,750 U/ml, about 1,500 to 4,500 U/ml, about 2,000 to 6,500 U/ml, about 2,500 to 5,500 U/ml, about 3,000 to 15,000 U/ml, or between any two of the previously disclosed concentrations.

In some embodiments, the mutanolysin concentration in the microbial lysis reaction is between about 1 to 1000 U/ml, about 5 to 200 U/ml, 20 to 800 U/ml, about 30 to 700 Um′, about 40 to 600 U/ml, about 50 to 500 U/ml, about 60 to 400 U/ml, about 70 to 300 U/ml, about 80 to 200 U/ml, about 90 to 100 U/ml, or between any two of the previously disclosed concentrations.

In some embodiments, the lysostaphin concentration in the microbial lysis solution is between about 500 to 50,000 U/ml, about 250 to 10,000 U/ml, 425 U to 8,000 U/ml, about 300 to 6,000 U/ml, about 400 to 5,000 U/ml, about 1,000 to 4,750 U/ml, about 1,500 to 4,500 U/ml, about 2,000 to 6,500 U/ml, about 2,500 to 5,500 U/ml, about 3,000 to 15,000 U/ml, or between any two of the previously disclosed concentrations.

In some embodiments, the lysostaphin concentration in the microbial lysis reaction is between about 1 to 1000 U/ml, about 5 to 200 U/ml, 20 to 800 U/ml, about 30 to 700 Um′, about 40 to 600 U/ml, about 50 to 500 U/ml, about 60 to 400 U/ml, about 70 to 300 U/ml, about 80 to 200 U/ml, about 90 to 100 U/ml, or between any two of the previously disclosed concentrations.

In some embodiments, one or more salts are added to the microbial lysis solution. In some embodiments, the concentration of the monovalents salts is between about 50 mM and 6 M, about 150 mM and 5 M, about 350 mM and 4.5 M, about 550 mM and 4 M, about 900 mM and 3.75 M, about 1 M and 3.5 M, or between any two of the previously disclosed concentrations. In some embodiments, the salt comprises one or more monovalent salts. By way of example, but not by way of limitation, in some embodiments, the monovalent salt is one or more of NaCl, KCl, and/or LiCl.

In some embodiments, the salt concentration in the microbial lysis reaction is between about 50 mM and 800 mM, about 100 mM and 700 mM, about 200 mM and 600 mM, about 300 mM and 500 mM, and about 350 mM and 450 mM, or between any two of the previously disclosed concentrations.

In some embodiments, the one or more monovalent salts is stored in dry or pelleted form, where upon re-suspension of the respective salt, the salt reaches the concentrations identified above.

In some embodiments, an enzymatic reaction time is between about 1-60 minutes, about 5-55 minutes, about 10-45 minutes, about 15-40 minutes, about 20-35 minutes, or about 25-30 minutes.

In some embodiments, DNA contaminants in the enzymatic reaction are removed or rendered non-amplifiable or unamplifiable. In some embodiments, removal of DNA is achieved using ion exchange resins.

In some embodiments, at least one DNA intercalating dye is added to the microbial lysis solution. In some embodiments, the DNA intercalating dyes are dyes that create a covalent bond to both DNA strands after activation with a light source of the appropriate wavelength and dosage. Without wishing to be bound by theory, in some embodiments, the covalent bond renders at least some of the DNA present in the sample unamplifiable. By way of example, but not by way of limitation, in some embodiments, the DNA intercalating dye include, but are not limited to, ethidium monoazide (EMA) and propidium monoazide (PMA).

In some embodiments, the concentration of the DNA intercalating dye in the microbial lysis solution is between about 0.01 μM to 1.0 μM, about 0.1 μM to 0.9 μM, 0.2 μM to 0.8 μM, about 0.3 μM to 0.7 μM, or about 0.4 μM to 0.6 μM, or between any two of the previously disclosed concentrations.

In some embodiments, the microbial lysis solution also includes one or more nucleases. In some embodiments, the nucleases are neutralized prior to usage of the microbial lysis solution. The exact nucleases used depend on the downstream sequences of interest. By way of example, but not by way of limitation, in some embodiments, the nucleases are selected from, but not limited to, EcoRI, HindIII, SaiI, HhaI, DdeiI, RsaI, Sau3AI and MspI.

In some embodiments, the microbial lysis solution includes one or more detergents. In some embodiments, the detergents or surfactants are non-ionic. Detergents and surfactants, include, but are not limited to BigCHAP, Deoxy BigCHAP, Brij 35, Brij 58P, Cymal-1, Cymal-2, Cymal-5, Cymal-6, Decyl-β-maltopyranoside, n-Dodecyl- -D-maltoside, n-Hexadecyl-β-D-maltoside, Undecyl-β-D-maltoside, Decyl-β-D-1-thiomaltopyranoside, Octyl-β-D-glucopyranoside, Decyl-β-D-1-thioglucopyranoside, Octyl-β-Dthioglucopyranoside, Digitonin, Dimethyldecylphosphine oxide (APO-10), Dodecyldimethylphosphine oxide (APO-12), IGEPAL CO-520, IGEPAL CO-630, and IGEPAL CO-720, N-Octanoyl-N-methylglucamine(MEGA-8), N-nonanoyl-N-methylglucamine(MEGA-9), N-Decanoyl-N-methylglucamine(MEGA-10), nonidet P40-substitute, Pluronic F-68, saponin, thesit, Triton X-100, Triton X-1 14, TWEEN 20, TWEEN TWEEN 80, sodium cholate, Sodium deoxycholate, sodium glycocholate, sodium taurocholate, sodium taurodeoxycholate, N-1-lauroylsarcosine, lithium dodecyl sulfate, sodium dodecyl sulfate (SDS), hexadecyltrimethyl ammonium bromide (CTAB), trimethyl(tetradecyl) ammonium bromide (TTAB), ASB-14(amidosulfobetaine-14), ASB-16(amidosulfobetaine-16), C7BzO, CHAPS, CHAPSO, EMPIGEN BB, 3-(N,N-Dimethyloctylammonio) propanesulfonate inner salt (SB3-8), 3-(decyldimethylammonio)-propanesulfonate inner salt (SB3-10), 3-(dodecyldimethylammonio)-propanesulfonate inner salt (SB3-12), 3-(N,N-dimethylmyristylammonio)-propanesulfonate(SB3-14), 3-(N,N-dimethylpalmitylammonio)-propanesulfonate(SB3-16), 3-(N,N-dimethyloctadecylammonio)-propanesulfonate(SB3-18), 3-(1-pyridinio)-1-propanesulfonate (NDSB 201), and 3-(benzyldimethylammonio) propanesulfonate (NDSB 256).

In embodiments, the concentration of the non-ionic surfactants required for lysis as found in the reaction is between 0.1-1%, is between 0.5-5%, is between 1%-10%, between 5%-50%, or between 10%-90%.

In some embodiments, the detergent is a zwitterionic detergent. In some embodiments, the zwitterionic detergent is from the sulfobetaine families. By way of example, but not by way of limitation, in some embodiments, sulfobetaine detergents include, but are not limited to, N-Decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, N-Decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, N-Dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, N-Hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, N-Octadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, and 3-[N,N-Dimethyl(3-myristoylaminopropyl)ammonio]propanesulfonate.

In some embodiments, the detergents are a non-ionic detergent from the glucopyranoside family. By way of example, but not by way of limitation, in some embodiments, non-ionic glucopyranoside detergents include, but are not limited to, 3-acetylumbelliferyl b-D-glucopyranoside, N-amyl b-D-glucopyranoside decyl b-D-thioglucopyranoside, n-dodecyl b-D-glucopyranoside, hexadecyl b-D-glucopyranoside, hexyl b-D-glucopyranoside, methyl a-D-glucopyranoside, octyl b-D-glucopyranoside, and phenyl-a-D-glucopyranoside.

In some embodiments, the detergent is a cationic detergent. By way of example, but not by way of limitation, in some embodiments, cationic detergents include, but are not limited to, alkyltrimethylammonium bromide, hexadecyltrimethylammonium bromide, hexadecylpyridinium bromide, myristyltrimethylammonium bromide, benzyldodecyldimethylammonium bromide, hexadecyl(2-hydroxyethyl)dimethylammonium, hexadecylpyridinium chloride, hexadecyltrimethylammonium chloride, or tetrakis(decyl)ammonium bromide. In some embodiments, the concentration of cationic detergents is between about 1-100× critical micelle concentration (CMC).

In some embodiments, a single detergent from the sulfobetaine and glucopyranoside family is added to the microbial lysis solution. In some embodiments, one or more detergents from the sulfobetaine family and the glucopyranoside family are added to the microbial lysis solution. Additionally, or alternatively, in some embodiments, the microbial lysis solution includes one or more cationic detergents. By way of example, but not by way of limitation, in some embodiments, cationic detergents include alkyltrimethylammonium bromide, amprolium hydrochloride, benzalkonium chloride, benzyldimethyldodecylammonium chloride, benzyldimethyltetradecylammonium chloride, benzyldodecyldimethylammonium bromide, cetylpyridinium chloride, cetyltrimethylammonium bromide, dimethyldioctadecylammonium bromide, dodecylethyldimethylammonium bromide, dodecyltrimethylammonium bromide, ethylhexadecyldimethylammonium bromide, hexadecylpyridinium bromide, hexadecylpyridinium chloride, hexadecyltrimethylammonium bromide, methylbenzethonium chloride, myristyltrimethylammonium bromide, oxyphenonium bromide, tetraheptylammonium bromide, tetrakis(decyl)ammonium bromide, tetrakis(decyl)ammonium bromide, and tricaprylylmethylammonium chloride.

In some embodiments, the concentration of the individual detergent is dependent on the critical micelle concentration (CMC) of the specific detergent in the microbial lysis reaction. In some embodiments, each detergent concentration in the microbial lysis solution is between about 10 to 1 1,000, about 25 to 12,500, about 50 to 8,000, about 75 to 7,000, about to 8,500, or about 98 to 6,750 times the CMC. In some embodiments, the detergent concentration in the microbial lysis solution is between about 100 to 5,000, about 125 to 9,000, about 200 to 8,000, about 400 to 7,000, or about 500 to 6,000 times the CMC.

In some embodiments, the detergent concentration in the microbial lysis solution is between about 100 to 1000, about 200 to 900, about 300 to 800, about 400 to 700, or about 500 to 600 times the CMC. In some embodiments, each detergent concentration in the microbial lysis reaction is between about 0.1 to 100, about 1.0 to 90, about 10 to 80, about 20 to 70, about 30 to 60, or about 40 to 50 times the CMC.

In some embodiments, the detergents (either as a group or individually, or any combination thereof) are stored in dry or pelleted form, where upon re-suspension of the respective detergent, the detergent reaches the concentrations identified above.

In some embodiments, the microbial lysis solution includes one or more metal chelators. By way of example, but not by way of limitation, in some embodiments, metal chelators include, but are not limited to, ethylene-glycol-tetra acetic acid (EGTA) and ethylenediaminetetraacetic acid (EDTA). In some embodiments, the concentration of the metal chelators in the microbial lysis solution is between about 50 mM to 1.0 M, about 100 mM to 0.75 M, about 110 mM to 500 mM, about 125 mM to 500 mM, about 125 mM to 450 mM, or between any two of the previously disclosed concentrations. In some embodiments, the concentration of the metal chelators in the microbial lysis reaction is between about 5 mM to 250 mM, about 10 mM to 100 mM, about 15 mM to 90 mM, about 20 mM to 80 mM, about 125 mM to 450 mM, or between any two of the previously disclose concentrations.

In some embodiments, the metal chelators are stored in dry or pelleted form, where upon re-suspension of the metal chelators, the metal chelators reach the concentrations identified above.

In some embodiments, the microbial lysis solution includes one or more reducing agents. By way of example, but not by way of limitation, in some embodiments, the reducing agent is 2-mercaptoethanol or dithiothreitol. In some embodiments, the concentration of the reducing agent in the microbial lysis solution is between about 10 mM to 20 M, about 15 mM to 15 M, about 50 mM to 14 M, about 100 mM to 14 M, or about 1 10 mM to 15 M, or between any two of the previously disclosed concentrations.

In some embodiments, the concentration of the reducing agent in the microbial lysis reaction is between about 1 mM to 100 mM, about 10 mM to 90 mM, about 20 mM to 80 mM, about 30 mM to 70 mM, about 40 mM to 60 mM, or about 45 mM to 55 mM, or between any two of the previously disclosed concentrations.

In some embodiments, the reducing agents are stored in dry or pelleted form, where upon re-suspension of the respective reducing agent, the reducing agent reaches the concentrations identified above.

In some embodiments, the microbial cell lysis reaction is performed at a pH below about 9. In some embodiments, the microbial cell lysis reaction is performed at a pH between about 6 to 9.

In some embodiments, the microbial cell lysis reaction is performed at about a neutral pH. In some embodiments, the microbial cell lysis methods disclosed herein, lead to the release of high molecular weight microbial DNA. Without wishing to be beyond by theory, in some embodiments, the microbial cell lysis methods disclosed herein lead to reduced shearing of microbial genetic materials during the microbial cell lysis and promote the presence of high molecular weight microbial DNA in the lysis solution. In some embodiments, high molecular weight microbial DNA is between about 2 kbp to 200 kbp, about 10 kbp to 190 kbp, about 20 kbp to 180 kbp, about 30 kbp to 170 kbp, about 40 kbp to 160 kbp, about 50 kbp to 150 kbp, about 60 kbp to 140 kbp, about 70 kbp to 130 kbp, about kbp to 120 kbp, or about 90 kbp to 110 kbp.

Isolation of Microbial Genomic Material

Having lysed the microbial content of the blood-based mixture, in some embodiments it is preferred to isolate or purify the microbial genomic-DNA (herein ‘gDNA’) from the non-DNA components of the sample. In contrast to the majority of current methods employing the addition of chaotropic salts to achieve the same, our preferred method entails the use of anion exchange resins for capturing free microbial gDNA and washing away non-DNA components from the system. Upon elution, and in some embodiments, the isolated gDNA has the advantage of being of sufficient purity such that it does not need to be diluted prior to downstream enzymatic amplification.

In some embodiments, after microbial cell lysis, the microbial genetic material is isolated and/or purified. In some embodiments, the genetic material isolated and/or purified is RNA or DNA. In some embodiments, the DNA is single stranded DNA (ssDNA) or double stranded DNA (dsDNA).

In some embodiments, microbial genetic material is isolated by contacting the microbial lysis reaction solution with anion exchange materials packed into columns, wherein the anion exchange material is used for the adsorption and subsequent elution of microbial genetic material. In some embodiments, a solution of known ionic strength and pH enable binding of nucleic acids to the anion exchange column and enable lesser-bound contaminants to be washed away. By way of example, but not by way of limitation, in some embodiments, conditions for selectively binding microbial genetic material with anion exchange materials include contacting the microbial lysis reaction solution with anion exchange in one or more of the following conditions: the contacting reaction is performed at a pH of between about 6 to 9, about 4.5 to 7, or about 8 to 9.5, and the contacting reaction has a monovalent salt concentration of between about 100 mM to 750 mM, about 450 mM to 1.75 M, or about 50 mM to 350 mM. The bound genetic material may then be eluted after contaminants have been removed. In some embodiments, an anion exchange resin is used to capture/immobilize microbial genomic material. In some embodiments, an anion exchange resin is one or more weak anion-exchange resins (WAX). Examples of WAX include, but are not limited to, carboxymethyl (CM), diethylaminopropyl (ANX), diethylethanolamine (DEAE), Amberlite Ira67, Purolite A847, Amberlite Ira96, Amberlite IRA96SB, Dowex Marathon WBA, Dowex Upcore Mono WB-500, Purolite A835, Dowex Monosphere 77, and Dowex Monosphere 66. In some embodiments, the WAX resin contains a tertiary amine functional group.

In some embodiments, an anion exchange resin is one or more strong anion-exchange resins (SAX). Examples of SAX include, but are not limited to, -0-CH₂—CHOH—CH₂-0-CH₂—CHOH—CH₂—N⁺(CH₃)₃, Amberjet Up4000, Amberjet 9000 OH, Amberlite FPA40 CI, and Dowex Upcore Mono MA-600. In some embodiments, a SAX based resin contains a quaternary amine functional group.

In some embodiments, the anion exchange resin is a combination of WAX and SAX.

In some embodiments, the form of the anion exchange resin is selected from fibers, membranes, sorbents, gels, and filter paper. In some embodiments, the sample with the lysed eukaryotic cells is passed through or contacted with the anion exchange resin. In some embodiments, the anion exchange resin is in a solution.

In some embodiments, the anion exchange resin is conjugated to a support substrate. Examples of a support substrate include, but are not limited to, a particle, a bead, a surface, or a sphere. In some embodiments, the support substrate is magnetic, e.g., a magnetic particle or bead. In some embodiments, the anion exchange resin is conjugated to a support substrate is in a solution.

In some embodiments, the support substrate comprises silica, glass, metal, polystyrene-based material, cellulose-based material, agarose-based material, dextran-based material, methacry late-based material, sepharose-based material, or a combination thereof. In some embodiments, the support substrate is porous.

In some embodiments, the support substrate is a bead or sphere has a diameter between about 10 to 100 μm, between about 20 to 90 μm, between about 30 to 80 μm, between about 40 to 70 μm, or between about 50 to 60 μm.

In another embodiment, the support substrate is a bead or sphere have a diameter between about 0.1 to 10 μm, between about 1.0 to 9.0 μm, between about 2.0 to 8.0 μm, between about 3.0 to 7.0 μm, or between about 4.0 to 6.0 μm.

In some embodiments, the microbial lysis reaction is incubated with the anion exchange resin between about 0.1 to 10 minutes, between about 2 to 9 minutes, between about 3 to 8 minutes, between about 4 to 7 minutes, or between about 5 to 6 minutes. In some embodiments, the microbial lysis reaction is incubated with the anion exchange resin between about 10 to 30 minutes, between about 12 to 28 minutes, between about 15 to 25 minutes, between about 18 to 23 minutes, or between about 19 to 22 minutes. In some embodiments, the microbial lysis reaction is incubated with the anion exchange resin for less than 1 minute.

In some embodiments, the microbial lysis reaction is incubated with the anion exchange resin between about 0.01 to 10 minutes, about 0.1 to 9 minutes, 1 to 8 minutes, about 2 to 7 minutes, 3 to 6 minutes, or about 4 to 5 minutes beyond that which is required to lysis the microbial cells.

In some embodiments, the anion exchange resin is permanently immobilized on the support substrate. In some embodiments, the immobilized anion exchange resin is contacted and/or incubated with the mixture and then the mixture is removed.

In some embodiments, at least one anion exchange resin conjugated to a support substrate, e.g., a bead or a particle (e.g., a microparticle), is contacted and/or incubated with the mixture. In some embodiments, after contacting and/or incubation with the microbial lysis reaction, the anion exchange resin conjugated to a support substrate is removed from the microbial lysis reaction. In another embodiment, after contacting and/or incubation with the microbial lysis reaction, the anion exchange resin conjugated to a support substrate is immobilized and the microbial lysis reaction is removed. By way of example, but not by way of limitation, in some embodiments, the anion exchange resin conjugated to a support substrate is selectively immobilized when the support substrate is a magnetized or metal bead and the magnetized or metal bead is exposed to a magnet or magnetic field.

In some embodiments, the beads or particle are packed into a column. In some embodiments, the beads or particle are free floating form.

In some embodiments, the anion-exchange-microparticles is a weak anion exchange material bound to magnetizable microspheres or microparticles. In some embodiments, the anion-exchange-microparticles is a strong anion exchange material bound to magnetizable microspheres.

In some embodiments, the anion-exchange-microparticles is a weak anion exchange material bound to porous agarose based-microspheres. In some embodiments, the anion-exchange-microparticles is a strong anion exchange material bound to porous agarose based-microspheres.

In some embodiments, after binding the microbial genetic material to the anion-exchange-microparticles, the anion-exchange-microparticles are washed using a wash buffer or wash solution.

In some embodiments, the pH of the wash solution is between about 7 to 11, about 8.5 to 10, or about 8 to 9.5. In some embodiments, the solution has a salt concentration of between about 0 mM to 1 M, 50 mM-900 mM, 100 mM-800 mM, or about 200 mM −600 mM.

In some embodiments, the wash solution includes one or more surfactants. By way of example, but not by way of limitation, in some embodiments, surfactants include, but are not limited to, Tween and Triton-X. In some embodiments, the Tween and/or Triton-X concentration is between about 0.01% to 1.0% (v/v), about 0.1% to 0.9% (v/v), about 0.2% to (v/v), about 0.3% to 0.7% (v/v), or about 0.4% to 0.6% (v/v). In some embodiments, the wash solution includes one or more detergents. By way of example, but not by way of limitation, in some embodiments, detergents include, but are not limited to, zwitterionic detergents. In some embodiments, the zwitterionic detergent concentration is between about 0.1× to 350× CMC, about 1.0× to 300× CMC, about 10× to 250× CMC, about 50× to 200× CMC, or about 100× to 150× CMC.

In some embodiments, the methods for isolating the microbial DNA includes an elution step. In some embodiments, competition of the isolation process is facilitated by eluting or removing the DNA off of the anion-exchange-microparticles.

In some embodiments, the pH of the elution buffer is between about 12 to 13.5. The use of an elution buffer with a pH greater than about 12 is not commonly used in the art.

In some embodiments, the elution buffer comprises of a buffering agent such as sodium phosphate or potassium phosphate. In some embodiments, the concentration of sodium phosphate or potassium phosphate is between about 0.01 M to 1 M, about 0.1 M to 1.8 M, about 0.4 M to 1.6 M, about 0.8 M to 1.4 M, or about 1.0 M to 1.2 M. In some embodiments, no buffering agent is required.

Additionally, or alternatively, in some embodiments, the elution buffer comprises sodium hydroxide or potassium hydroxide. In some embodiments, the concentration sodium hydroxide or potassium hydroxide is between about 10 to 500 mM, about 30 to 450 mM, about 50 to 400 mM, about 70 to 350 mM, about 90 to 300 mM, about 1 10 to 250 mM, or about 130 to 200 mM.

In some embodiments, the elution buffer also includes one or more monovalent salts. By way of example, but not by way for limitation, in some embodiments, monovalent salts include, but are not limited to, NaCl, KCl and LiCl.

In some embodiments, the concentration of the one or more monovalent salts in the elution buffer is between about 0 mM to 200 mM, about 25 mM to 175 mM, about 50 mM, to 150 mM, about 75 mM to 125 mM, or about 90 mM to 110 mM. The use of an elution buffer with monovalent salt concentrations less than about 200 mM is not commonly used in the art. In some embodiments, the elution buffer does not contain any monovalent salts.

In some embodiments, no additional purification or desalting is required after eluting the genomic material from the anion-exchange resin.

In some embodiments, the gDNA is concentrated and/or purified using a size exclusion membrane following elution from the anion exchange resin. In some embodiments, the gDNA is concentrated and/or purified by applying one or more binding, wash, and/or elution steps to the anion exchange resin. In some embodiments, the concentration and/or purification comprises one or more of the following: (i) one or more binding steps; one or more washing steps; and one or more elution steps. Those skilled in the art will be to modify the process to meet purity and volume restrictions as required for optimal operation. Notwithstanding the above, this process, as well as the process for preparing the reagents, is illustrated in detail in WO2016044621A1.

Enzymatic Amplification of the Microbial Genomic Material

In some embodiments, it is preferred to enzymatically amplify the microbial genetic material (microbial gDNA). In some embodiments, the isolated microbial genetic material is subject to amplification. In some embodiments, the genetic material amplified is RNA or DNA. In some embodiments, the DNA is single stranded DNA (ssDNA) or double stranded DNA (dDNA). In some embodiments, the DNA is ribosomal DNA (rDNA). In some embodiments, the DNA is a gene. In some embodiments, the DNA is a plasmid. In some embodiments, microbial genetic material specific to a species or genus of microorganisms is amplified.

In some embodiments, enzymatic amplification can be achieved either through isothermal amplification or thermal-cycling amplification processes. In some embodiments, polymerase chain reaction, or PCR, is the preferred method of enzymatic amplification which is a well-known method of thermal-cycling based enzymatic amplification.

In some embodiments, a single amplification reaction is performed, e.g., the gDNA is not split into more than one reaction. Without wishing to be bound by theory, this can increase sensitivity.

In some embodiments, the amplification reaction is single-plex, e.g., utilizes a single pair of PCR primers. In some embodiments, the amplification reaction is multi-plex, e.g., utilizes a multiple pair of PCR primers. In some embodiments, the amplification reaction includes an additional set of primers for either internal or external control purposes.

In some embodiments, the amplicon is greater than about 400 bp. In some embodiments, the amplicon is between about 400 to 4000 bp, about 700 to 3700 bp, about 1000 to 3400 bp, about 1300 to 3100 bp, about 1600 to 2700 bp, about 1900 to 2400 bp, or about 2100 to 2200 bp. In some embodiments, use of amplicons of the lengths disclosed above are advantageous for downstream processing (e.g., detection and identification of microbial genetic materials) in the methods disclosed herein.

In some embodiments, the amplified genetic material comprises a bacterial gene or plasmid that is conserved. In some embodiments, the amplified genetic material comprises a bacterial plasmid that is stable. In some embodiments, the amplified genetic material comprises a gene or plasmid that is specific to Borrelia. In some embodiments, the amplified genetic material comprises a gene or plasmid that allows for the identification of the genus Borrelia as well as individual species within the genus. In some embodiments, the amplified genetic material comprises a plasmid selected from BB147, cp9, cp26, cp32-1, cp32-3, cp32-4, cp32-6, cp32-7, cp32-8, cp32-9,1p5, 1p17,1p21,1p25A,1p25B,1p28-1A,1p28-1B,1p28-2, 1p28-3,1p28-4, 1p36, 1p38, 1p54, 1p56. In some embodiments, the amplified genetic material comprises a gene selected from OspA, OspB, OspC, fla, and omp66.

In some embodiments, the amplification product is purified. By way of example, but not by way of limitation, in some embodiments, a method for purifying the amplification product includes the reversible binding or absorption of the amplicon onto glass or silica fibers or particles in combination with chaotropic salts followed by their washing and elution. In some embodiments, purification methods include, but is not limited to, precipitation in an alcohol-based solutions (e.g., such as ethanol or isopropanol), contacting with anion exchange resins, or size exclusion filters. In some embodiments, the cleaning-up of the amplification product removes excess primers, dNTPs, salts and other components that may interfere with downstream processes.

In some embodiments, no purification process is required, and the amplification product/solution can be used as is in downstream processes.

In some embodiments, the microbial genetic material is amplified by PCR and the number of PCR cycles are modified to adjust for sample input volume, sample type, and/or microbial load assessments. In some embodiments, the microbial genetic material is amplified by isothermal amplification and the amplification times are modified to adjust for sample input volume, sample type, and/or microbial load assessments.

Notwithstanding the above, this process, as well as the process for preparing the reagents, is illustrated in detail in WO 2016/044621A1.

Detection of Amplified Genomic Material

In some embodiments, the amplified genetic material is detected, and/or identified, and/or characterized by quantitative PCR. In some embodiments, the amplified genetic material is detected, and/or identified, and/or characterized by microarray analysis. In some embodiments, the amplified genetic material is detected, and/or identified, and/or characterized by DNA sequencing. In some embodiments, the amplified genetic material is detected, and/or identified, and/or characterized by melting curve analysis. In some embodiments, the amplified genetic material is detected, and/or identified, and/or characterized by mass spectrometry. Each of these techniques is commonly known to those of skill in the art.

In some embodiments, DNA Invading Artificial Nucleic Acids (DIANAs) are used detect and/or identify, and/or characterize microbial genetic materials. In some embodiments, the process of invasion, in contrast to hybridization, specifically targets double stranded DNA, or regions within a single-stranded DNA that are double stranded, negating the need to fully denature double stranded DNA (see, e.g., Egholm et ah, Nucleic Acids Res. 23(2): 217-222 (Jan. 25, 1995).

In some embodiments, the DIANAs take the form of a specialized type or class of Peptide Nucleic Acids (PNAs). In some embodiments, the DIANAs are not limited to a specific class of PNAs. In some embodiments, the DIANAs take the form of a specialized type or class of Locked or Bridged Nucleic Acids (LNAs and/or BNAs). In some embodiments, DIANAs that locally invades duplex DNA has the required affinity and sequence specificity to be used in the methods disclosed herein.

In some embodiments, PNA oligomer based DIANAs have a chiral stereo-center at the gamma-position of the backbone (also known as γPNA). A PNA oligomer that is pre-oriented structurally into a right-handed helix is energetically favored to perform duplex DNA invasion. In some embodiments, the microbial DNA is detected using γPNA as taught in WO 2013/176992, the contents of which are incorporated by reference in its entirety. In some embodiments, use of DIANAs is advantageous for long amplicons (e.g., amplicons between about 400 to 4000 bp).

In some embodiments, each DIANA targets a specific sequence found in microbial genetic material (e.g., DNA or RNA) from a single microbial species, e.g., a specific Borrelia species. In some embodiments, each DIANA targets a specific sequence found in microbial genetic material (e.g., DNA or RNA) from a group of microorganisms, e.g., multiple Borrelia species, e.g., broad-Borrelia. In some embodiments, each DIANA targets a single strain of microorganisms. In some embodiments, each DIANA targets a more than one strain of microorganisms. In some embodiments, each DIANA targets a number of species, from different genus of microorganisms. In some embodiments, each DIANA targets a number of species, from different the same genus of microorganisms. In some embodiments, multiple DIANA sequences are used to a strain, species, or genus of microorganisms.

In some embodiments, the specific microbial genetic material (e.g., DNA or RNA) is amplified microbial genetic material.

In some embodiments, the DIANAs are modified to contain a binding moiety. In some embodiments, the binding moiety binds the DIANA to a solid substrate. In some embodiments, the binding DIANA to a solid substrate is useful for separation or washing steps downstream. By way of example, but not by way of limitation, in some embodiments, the binding moieties include, but are not limited to, non-covalent binding moieties (e.g., such as biotin, digoxin, digitoxin) or covalent binding moieties (e.g., COOH group, NHS-ester group, malemide chemistry, and Click chemistry).

In some embodiments, the binding moiety is spaced from the DIANA probe by one or more linkers. In some embodiments, the linker is a single molecule. In some embodiments the linker is comprised of a chain of multiple individual molecules, either linear or branched, that are combined to create a single linker molecule.

In some embodiments, the linker is selected from the group consisting of: (ethylene) glycol, di(ethylene)glycol, tri(ethylene)glycol, poly(ethylene)glycol, carbon linker, amino acids, a silane-based linker, or any combination thereof. In some embodiments, the linker serves to distance the DIANA tagged DNA fragment from the surface of the solid phase substrate to which the DIANA is bound to.

In some embodiments, the linker is 4 atoms in length or greater. In some embodiments, the linker is 4-200 atoms in length.

In some embodiments, one or more binding moieties are used along a single linker. In some embodiments, two or more binding moieties along a single linker, wherein each linker has 1 or more binding moieties and wherein each binding moiety is attached to a different location along the oligomer. In some embodiments, multiple binding moieties increase the surface binding kinetics and/or yield and/or efficiently, and/or strength.

In some embodiments, the DNA amplicon is first tagged with one or more DIANAs and prior to capturing the hybrid complex onto a solid-phase surface.

In some embodiments, the solid-phase surface is a bead, nanoparticle, microparticle or flat substrate. In some embodiments, the solid-phase surface is further chemically modified to facilitate binding of the DIANA to it.

In some embodiments, capturing a target amplicon and immobilizing it onto the solid-phase surface occurs in individuals wells on system (e.g., a plate or a chip).

In some embodiments, a well is activated with a single DIANA oligomer. In some embodiments, a well is activated with more than one DIANA probe for a single pathogen. In some embodiments, one or more probes may be used for multiple pathogens.

In some embodiments, the location (well number/position) will yield the information as to which target was captured (e.g., due to the presence of a DIANA probe). In some embodiments, a combination of detected color (e.g., when fluorescence is used as the optical detection modality) and location can be used to decipher which target was captured.

In some embodiments, ssDNA are utilized rather than dsDNA. In some embodiments, ssDNA are created from dsDNA via denaturing protocols or through an asymmetric amplification process prior to DIANA tagging of the DNA molecule.

In some embodiments the DNA is entirely in duplex form. In some embodiments, the DNA is locally in duplex form.

In some embodiments, the incubation of DIANAs and the microbial genetic material (e.g., amplified microbial DNA) is at a temperature between about 20° C. to 65° C. In some embodiments, the incubation of DIANAs and the microbial genetic material is at a temperature between about 25° C. to 65° C. In some embodiments, the incubation of DIANAs and the microbial genetic material is at a temperature between about 30° C. to 65° C. In some embodiments, the incubation of DIANAs and the microbial genetic material is at a temperature between about 37° C. to 65° C. In some embodiments, the incubation of DIANAs and the microbial genetic material is at a temperature between about 45° C. to 65° C. In some embodiments, the incubation of DIANAs and the microbial genetic material is at a temperature between about 55° C. to 65° C. In some embodiments, the incubation of DIANAs and the microbial genetic material is at a temperature of about 25° C., 26° C., 27° C., 28° C., 29° C., 30° C., 31° C., 32° C., 33° C., 34° C., 35° C., 36° C., 37° C., 38° C., 39° C., 40° C., 41° C., 42° C., 43° C., 44° C., 45° C., 46° C., 47° C., 48° C., 49° C., 50° C., 51° C., 52° C., 53° C., 54° C., 55° C., 56° C., 57° C., 58° C., 59° C., 60° C., 61° C., 62° C., 63° C., 64° C., or 65° C. In some embodiments, the incubation of DIANAs and the microbial genetic material (e.g., amplified microbial DNA) is at a temperature between about 65° C. to 99° C., about 70° C. to 95° C., about 75° C. to 90° C., or about 80° C. to 85° C.

Provided herein are methods that provide for the invasion of DIANAs at the reduced temperatures of above 25° C. DIANAs in 10 minutes or less. As is described in more detail below, the use of invasion temperatures below 65° C. for invasion reactions lasting 10 minutes or less is new and advantageous.

In some embodiments, the invasion reaction last between about 0.1 to 5 minutes, about 1 to 10 minutes, about 5 to 30 minutes, or about 10 to 60 minutes. In some embodiments, the invasion reaction lasts less than 10 minutes, less than 9 minutes, less than 8 minutes, less than 7 minutes, less than 6 minutes, less than 5 minutes, less than 4 minutes, less than 3 minutes, less than 2 minutes, or less than 1 minute, for example, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, or 10 minutes.

By way of example, but not by way of limitation, in some embodiments, the DIANA invasion process includes DIANA oligomers that have between about 14 to 18 bases, wherein the lower invasion temperature is defined as about: T_(M)(DNA)+15° C. and the upper invasion temperature is 99° C. T_(M)(DNA) is defined as the melting temperature of a DNA oligomer with identical composition and sequence to the DIANA oligomer when placed in nearly identical solution conditions (electrolytes strength, buffer, pH, other additives, etc.).

By way of example, but not by way of limitation, in some embodiments, the DIANA invasion process includes using DIANA oligomers that are larger than 18 bases, wherein the lower invasion temperature is defined as about: T_(M)(DNA)+0.7° C.×(number of bases) and the upper invasion temperature is 99° C.

By way of example, but not by way of limitation, in some embodiments, the DIANA invasion process includes using DIANA oligomers that are smaller/shorter than 14 bases, wherein the lower invasion temperature is defined as about: T_(M)(DNA)+1.1° C.×(number of bases) and the upper invasion temperature is 99° C.

In some embodiments, the composition of the DIANA invasion solution is depicted in WO 2016/044621A1.

In some embodiments, the invasion solution includes a buffering agent. By way of example, but not by way of limitation, in some embodiments, the buffering agent includes, but is not limited to, tris, sodium-phosphate, and potassium phosphate.

In some embodiments, the concentration of the buffering agent is between about 1 mM to 500 mM, about 50 mM to 450 mM, about 100 mM to 400 mM, about 150 mM to 350 mM, or about 200 mM to 300 mM. In some embodiments, no buffering agent is required. In some embodiments, the pH of the invasion solution is between about pH 6 and about pH 9. In some embodiments, the invasion solution includes one or more monovalent salts.

In some embodiments, the monovalent salt is NaCl or KCl. In some embodiments, the concentration of monovalent salt is between about 1 mM to 150 mM, about 5 mM to 145 mM, about 15 mM to 130 mM, about 25 mM to 1 15 mM, about 35 mM to 100 mM, about mM to 85 mM, or about 55 mM to 70 mM. In some embodiments, the invasion solution contains no monovalent salts. The disclosed salt concentrations of the invasion assay are below the salt concentration used in standard hybridization assays.

In some embodiments, the invasion solution include one or more surfactants. In some embodiments, the surfactant reduces non-specific binding. By way of example, but not by way of limitation, surfactants include, but are not limited to, Tween-20, or TritonX-100. In some embodiments, the concentration of the surfactant in the invasion solution is between about 0.01% to 1.0% (v/v), about 0.1% to 0.9% (v/v), about 0.2% to 0.8% (v/v), about 0.3% to 0.7% (v/v), or about 0.4% to 0.6% (v/v).

In some embodiments, the invasion solution includes components to vary the excluded volume (e.g., crowding agents). By way of example, but not by way of limitation, crowding agents include, but are not limited to, poly-ethylene glycol (PEG), PEG-200, PEG-250, PEG-300, PEG-400, PEG-500, PEG-750, PEG-1,000, PEG-9,500, PEG-2,000, PEG-4,000, PEG-5,000, PEG-6,000, PEG-8,000, PEG-10,000, PEG-12,000, PEG-13,000, PEG-dextrans (DX), polyvinyl-alcohols (PVA), Ficolls (FC), DX-1,000, DX-5,000, DX-12,000, DX-50,000, DX-80,000, PVA 89k-98k, PVA 85k-124k, PVA 130k, PVA 31k-50k, PVA 50k-80k, PVA 70k-100k, PVA 90k-120k, PVA 170k-250k, PVA 61k, PVA 31k, PVA 130k, PVA 67k, PVA 27k, PVA 25k, FC-400, FC-70, FC-40, glycerol, glucose, and sucrose. In some embodiments, the concentration range of the crowding agent in the invasion solution is between about 1% to 20% (v/v), about 3% to 17% (v/v), about 6% to 14% (v/v), or about 9% to 11% (v/v) of the total volume of invasion solution. In some embodiments, the invasion solution included one or more DNA denaturants. By way of example, but not by way of limitation, DNA denaturants include, but are not limited to, DMSO, formamide, and betaines.

In some embodiments, the invasion solution also includes DMSO, formamide, betaines, or a combination thereof. In some embodiments, the DMSO and/or formamide are between about 1% to 30% (v/v), about 5% to 25% (v/v), about 10% to 20% (v/v), or about 14% to 16% (v/v) of the total volume of invasion solution. In some embodiments, the concentration of the betaines in the invasion buffer is between about 0.1 M and 2.5 M, about 0.5 M and 2.0 M, or about 1.0 M and 1.5 M.

In some embodiments, the invasion solution has a pH of about 10 or more. In some embodiments, an invasion solution with a pH greater than about 10 is conducive to DNA denaturing or destabilization.

Washing

In some embodiments, a washing step is performed after DIANA invasion. In some embodiments, the wash step reduces non-specific binding. In some embodiments, the wash uses high temperature wash solutions. In some embodiments, the temperature of the wash solution is between about 60° C. and 99° C., about 65° C. and 95° C., about 70° C. and 90° C., or about 75° C. and 85° C., or between 20° C. to 65° C. The composition of the preferred DIANA wash buffer is depicted in WO 2016/044621A1.

In some embodiments, the wash buffer comprises one or more of the following: 1) monovalent salt, e.g., as NaCl or KCl, at between about 50 to 650 mM, about 100 to 600 mM, about 150 to 550 mM, about 200 to 500 mM, about 250 to 450 mM, or about 300 to 400 mM; 2) buffered to a near neutral pH, for example between about 6-9; and 3) surfactants, e.g., Tween-20 or Triton X-100 at between about 0.1% to 1.0% (v/v), about 0.2% to 0.9% (v/v), about 0.3% to 0.8% (v/v), about 0.4% to 0.7% (v/v), or about 0.5% to 0.6% (v/v). In some embodiments, the wash buffer is heated.

In some embodiments, the wash buffer includes one or more DNA destabilizing or denaturing agents, e.g., DMSO, betaines, and formamide. In some embodiments, the DMSO and/or formamide are between about 10% to 30% (v/v), about 15% to 25% (v/v), about 10% to 20% (v/v), or about 14% to 16% (v/v) of the total volume of invasion solution. In some embodiments, the concentration of the betaines in the invasion buffer is between about 0.1 M and 2.5 M, about 0.5 M and 2.0 M, or about 1.0 M and 1.5 M.

In some embodiments, the pH of the wash buffer is above 9.0 and includes between about 0 mM to 300 mM, about 50 mM to 250 mM, about 100 mM to 200 mM, or about 125 mM to 175 mM of monovalent salts and/or surfactants. In some embodiments, the pH of the wash buffer is below 9.0 and includes between about 0 mM to 800 mM, about 50 mM to 750 mM, about 100 mM to 700 mM, about 150 mM to 650 mM, or about 200 mM to 600 mM, about 250 mM to 550 mM, about 300 mM to 500 mM, or about 350 mM to 450 mM of monovalent salts and/or surfactants.

By way of example, but not by way of limitation, in some embodiments, the washing step comprises washing DIANA oligonucleotides that are sized between about 14 to 18 bases, wherein the lower wash temperature is defined as about: T_(M)(DNA)+20° C. and the upper wash temperature is 99° C.

In some embodiments, the preferred temperature for invasion and washing is dictated by the length of the DIANA probe, its base composition (i.e. GC content), and the conditions at which the reactions take place. Without wishing to be bound by theory, in some embodiments, the DIANA invasion reaction is rate limited by that which the duplex DNA region of interest can be effectively ‘opened’, thus exposing the nucleobases. As such, an increase in temperature is but one parameter which plays a role, which additive reagents also play a role. Further, with regards to washing conditions, and without wishing to be bound by theory, in some embodiments, the DIANA wash conditions are dependent on, as a minimum, the binding strength of the DIANA probe to the target DNA. As such, parameters such as temperature, electrolytes, pH, other additives, play a significant role in establishing the optimal condition.

By way of example, but not by way of limitation, in some embodiments, the washing step comprises washing DIANA oligomers that are sized between about 14 to 18 bases, wherein the lower wash temperature is defined as about: T_(M)(DNA)+20° C. and the upper wash temperature is 99° C.

By way of example, but not by way of limitation, in some embodiments, the washing step comprises washing DIANA oligonucleotides that are larger than 18 bases, wherein the lower wash temperature is defined as about: T_(M)(DNA)+0.9° C.×(number of bases) and the upper wash temperature is 99° C.

By way of example, but not by way of limitation, in some embodiments, the washing step comprises washing DIANA oligonucleotides that are smaller/shorter than 14 bases, wherein the lower wash temperature is defined as about: T_(M)(DNA)+1.25° C.×(number of bases) and the upper wash temperature is 99° C.

Low Temperature DIANA Invasion and Wash

Without wishing to be bound by theory, the process of invasion is similar to that of hybridization wherein binding is chiefly due to, but not limited to, Watson-Crick base-pairing rules. By indicating this, the intent is to highlight that a pre-requisite for invasion is ‘access’ to the nucleobases, which in the case of duplex DNA (either locally or universally and discussed below) is ‘hidden’ in most cases.

Without wishing to be bound by theory, the rate limiting step for DIANA invasion is the ability to open the duplex DNA thus making available the nucleobases for invasion. ‘Open’ does not necessarily mean that the DNA is denatured, but rather that what is known as DNA breathing is increased, where local, transient, bubbles are formed within the duplex DNA. As breathing increases these bubbles become (1) more frequent, (2) more common, (3) longer lived i.e. more stable, and (4) larger. DNA breathing is a natural, physical, process depicting the competing energetics of the negative sugar-phosphate backbone and the hydrogen bonds between the nucleobases and base-pair stacking interactions. DNA breathing may be unrelated to the presence or absence of DIANAs in the system.

Art known methods for DIANA invasion commonly described the use of temperatures at or below 37° C. At such temperatures, invasion was extremely slow—on the scale of hours. At even lower temperatures, moving towards ambient temperatures, DNA invasion becomes even slower. Cleary, a need exists for more rapid invasion in the field of rapid diagnostic technology.

Reaction conditions which enable rapid and highly efficient DNA invasion, in the 1-10-minute timeframe have recently been described. These methods are disclosed in WO 2016/044621A1. The methods disclosed in WO 2016/044621A1 can be useful at temperatures above about 65° C. (see section starting at para. [0248]).

Disclosed herein are methods for further reducing the invasion temperature to below 65° C., in certain conditions, while still meeting the sub-10 min (indeed the sub 5 min) timeframe. These methods employ the use of DIANA technology with predominantly single stranded DNA or RNA. This has not been previously described.

In some embodiments, the invasion can be accomplished at high speed at a reduced temperature in inherently duplex nucleic acid molecules in destabilizing conditions. Without wishing to be bound by theory, the conditions described herein are not meant to enable complete denaturization of the DNA template, but rather sufficient destabilization to enable a reduce temperature for invasion. The exact nature of these conditions are dependent on the reaction solution used with regards to denaturants and electrolyte concentrations as identified in WO 2016/044621A1 and described herein, in addition to the length of the duplex target.

In some embodiments, the invasion solution has a pH (either buffered or unbuffered) of about 10.2-12.2. In some embodiments, the pH is about 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.9, 11.0, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12.0, 12.1, or 12.2. In some embodiments, the pH is between 10.2 and 11.0. In some embodiments, the pH is between 10.5 and 11.5. In some embodiments, the pH is between 11.0 and 12.0. In some embodiments, the pH is 10.2 or above. In some embodiments, the pH is 10.5 or above. In some embodiments, the pH is 11.0 or above. In some embodiments, the pH is 11.5 or above. In some embodiments, the preferred pH is optimized for the specific data target, reaction additives, target length and GC composition, and preferred temperature range.

In some embodiments, a wash solution, used to remove non-specific binding of DIANAs to DNA, may likewise be used at temperatures between 25° C.-65° C. In some embodiments, the aforementioned wash solution has a pH (either buffered or unbuffered) of about 10.7-12.7. In some embodiments, the pH is about 10.7, 10.8, 10.9, 11.0, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12.0, 12.1, 12.2, 12.4, 12.4, 12.5, 12.6, or 12.7. In some embodiments, the pH is between 10.7 and 11.5. In some embodiments, the pH is between 11.0 and 11.8. In some embodiments, the pH is between 11.3 and 12.0. In some embodiments, the pH is between 11.7 and 12.7. In some embodiments, the pH is 10.7 or above. In some embodiments, the pH is 11.0 or above. In some embodiments, the pH is 11.5 or above. In some embodiments, the pH is 12.0 or above. In some embodiments, the preferred pH is optimized for the specific data target, reaction additives, target length and GC composition, DIANA length and preferred temperature range.

In other embodiments, a target DNA or RNA is predominantly single-stranded. In some embodiments, a double-stranded structure is induced locally to create the preferred conditions. While RNA is naturally single-stranded, DNA is naturally double-stranded. In some embodiments, double stranded DNA is processed to generate single stranded DNA. Processing steps include, but are not limited to enzymatic, chemical, or mechanical processing. Other processing methods are well known within the art.

Upon having in place single stranded DNA or RNA target molecules, local duplex, or hairpin, structures can be stabilized. This can be accomplished by increasing the electrolyte concentrations in the reaction mixture. In some embodiments, electrolytes are added to the invasion solution.

In some embodiments, monovalent salts are added to the invasion solution. In some embodiments, the monovalent salt is added at a concentration of above 50 mM. In some embodiments, the monovalent salt is added at a concentration of 100 mM or above. In some embodiments, the monovalent salt is added at a concentration of 200 mM or above. In some embodiments, the monovalent salt is added at a concentration of about 50 mM, 51 mM, 55 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM 125 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 175 mM, 180 mM, 190 mM, 200 mM, 225 mM, 250 mM, 275 mM, 300 mM, 325 mM, 350 mM, 375 mM, 400 mM, 450 mM, or 500 mM. In some embodiments, the monovalent salt is added at a concentration of from 51 mM-500 mM, from 51 mM-250 mM, from 51 mM-100 mM, or from 100 mM-200 mM.

In some embodiments, divalent salts are added to the invasion solution. In some embodiments, the monovalent salt is added at a concentration of above 5 mM. In some embodiments, the monovalent salt is added at a concentration of 7 mM or above. In some embodiments, the monovalent salt is added at a concentration of 10 mM or above. In some embodiments, the monovalent salt is added at a concentration of about 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, or 25 mM. In some embodiments, the monovalent salt is added at a concentration of from 6 mM-50 mM, from 6 mM-25 mM, from 6 mM-10 mM, or from 10 mM-20 mM.

In some embodiments, trivalent salts are added to the invasion solution. In some embodiments, the monovalent salt is added at a concentration of above 0.1 mM. In some embodiments, the monovalent salt is added at a concentration of 0.3 mM or above. In some embodiments, the monovalent salt is added at a concentration of 0.5 mM or above. In some embodiments, the monovalent salt is added at a concentration of about 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1.0 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 2.0 mM, or 2.5 mM. In some embodiments, the monovalent salt is added at a concentration of from 0.2 mM-1.0 mM, from 0.2 mM-0.7 mM, from 0.2 mM-0.5 mM, or from 0.5 mM-1.0 mM.

Detection of DIANA Binding

In some embodiments, detection of the binding of DIANAs to their respective target is through optical, chemical, electrical, or mechanical detection methods in a detection region. Method utilized for detection of the DIANAs to their respective target is depicted in WO 2016/044621A1.

In some embodiments, optical detection is through the use of fluorescence or luminescence.

In some embodiments, one or more detectable markers are positioned on the invading DIANAs. In some embodiments, the one or more detectable markers are positioned on the DNA amplicon captured via the immobilized oligonucleotide. In some embodiments, one or more detectable markers are positioned on a second oligonucleotide, which is universal to some or all potential targets.

By way of example, but not by way of limitation, in some embodiments, the detectable markers include, but are not limited to fluorescent dyes, quantum dots, horseradish peroxidase (HRP), luciferase, methoxycoumarin, dansyl, pyrene, Alexa Fluor 350, AMCA, Marina Blue dye, dapoxyl dye, dialkylaminocoumarin, bimane, hydroxycoumarin, cascade blue dye, Pacific Orange dye, Alexa Fluor 405, Cascade Yellow dye, Pacific Blue dye, PyMPO, Alexa Fluor 430, Fluorescein, Alexa Fluor 488, Oregon Green 488, BODIPY 493/503, Oregon Green 514, Alexa Fluor 514, Alexa Fluor 532, BODIPY TMR, Alexa Fluor 555, Alexa Fluor 546, BODIPY 558/568, Rhodamine Red dye, Alexa Fluor 568, BODIPY 581/591, Alexa Fluor 594, Texas Red dye, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, and Alexa Fluor 790.

By way of example, but not by way of limitation, detectable markers enabling indirect detection include, but are not limited to, digoxigenin (DIG), biotin, or dinitrophenyl.

In some embodiments, identification of the microbial species is through DNA amplicon labeling.

In some embodiments, the primers used in the amplification are labeled during with a detectable marker prior to beginning the amplification process.

In some embodiments, modified nucleotides that either contain a tag or are modified to enable the downstream conjugation of tags are used in the amplification process. By way of example, but not by way of limitation, tag-modified nucleotides include, but are not limited to, a nucleotide modified with a diethylaminocoumarin (DEAC), Cyanine 3 (Cy3), Cyanine 5 (Cy5), Fluorescein (FITC), Lissamine, R1 10, R6G, Tetramethylrhodamine (TAMRA), or Texas Red dye. Examples of a modified nucleotides enabling subsequent tagging would be, but are not limited to, a nucleotide modified with an Amino-digoxigenin (DIG), Biotin, or Dinitrophenyl (DNP).

In some embodiments, the labeling of the DNA amplicon is achieved through subsequent incubation with an intercalating dye. By way of example, but not by way of limitation, intercalating dyes include, but are not limited to, PicoGreen, OliGreen, RiboGreen, SYBR Gold, SYBR Green I, SYBR Green II, SYBR Safe, TOTO-1, YOYO-1, YOYO-3, POPO-1, BOBO-1, JOJO-1, POPO-3, LOLO-1, BOBO-3, YOYO-3, TOTO-3, SYTOX-Blue, SYTOX-Green, SYTOX-Orange, SYTOX-Red, and EtBr.

In some embodiments, the DNA amplicon is first tagged with one or more DIANAs and then the hybrid complex is captured onto the solid-phase surface.

In some embodiments, the DIANA is incubated with a solid surface prior to capturing the amplicon.

In some embodiments, the solid-phase surface is a bead, nanoparticle, microparticle or flat substrate. In some embodiments, the solid-phase surface is further chemically modified to facilitate binding of the DIANA to it.

In some embodiments, the detection region is the same region, e.g., in the same well, tube, or chamber, or in the same region on a fluidic cassette, where DIANA invasion/washing processes were conducted. In other embodiments, the detection region is a different same region from where DIANA invasion/washing processes were conducted.

In some embodiments, the methods described herein have a limit of detection (LOD) of between 1 CFU/100 ml-100 CFU/ml. In some embodiments, the methods described herein have a LOD of between 1 CFU/50 ml-50 CFU/ml. In some embodiments, the methods described herein have a LOD of between 1 CFU/10 ml-10 CFU/ml. In some embodiments, the LOD is less than 1 CFU/ml, less than 1 CFU/10 ml, or less than 1 CFU/100 ml.

In some embodiments, the methods described herein have a LOD of between 1 cell/100 ml-100 cell/ml. In some embodiments, the methods described herein have a LOD of between 1 cell/50 ml-50 cell/ml. In some embodiments, the methods described herein have a LOD of between 1 cell/10 ml-10 cell/ml. In some embodiments, the LOD is less than 1 cell/ml, less than 1 cell/10 ml, or less than 1 cell/100 ml.

In some embodiments, the volume of the sample affects the LOD of the method. By way of example, but not by way of limitation, an increase in the inputted sample-volume will allow for the detection of rarer microorganisms, increasing the sensitivity of the LOD measurement.

In some embodiments, all types of microorganisms have a similar LOD, whereas in other embodiments, individual LODs may vary.

In some embodiments, the limit of detection of microorganisms may not be measurable using the standard of CFU or Colony Forming Units per unit volume, as the microorganism may (1) not form colonies, or (2) may be uncultureable.

Quantification of Microbial Load

In some embodiments, the methods described herein comprise monitoring microbial, e.g., pathogen, load. This is useful, for example, in the context of measuring the load of a microbe or microbes in a subject over time, to monitor the course of infection, or to observe the response of the microbe to therapeutic intervention, e.g., antibiotics or antifungals. In some embodiments, the methods described herein provide is the ability to measure microbial load quantitatively, i.e., the methods provide a direct correlation between inputted pathogen load and signal output. In some embodiments, the methods described herein provide the ability to measure microbial load semi-quantitatively.

In some embodiments, the ability to measure microbial load is useful clinically, medically, or scientifically.

In some embodiments, the microbial load is measured over time, e.g., at multiple time points, e.g., at a first and second time point. In some embodiments, measuring microbial load at a first and second time point can allow the course of infection or response to treatment to be monitored in a subject. In some embodiments, an increase in microbial, e.g., pathogen, load indicates that the subject has an infection that is worsening. In some embodiments, an increase in microbial, e.g., pathogen, load indicates that the subject has an infection that is not improving. In some embodiments, no change in microbial, e.g., pathogen, load indicates that the subject has an infection that is not resolving. In some embodiments, if the subject is receiving treatment, e.g., with an antimicrobial, an increase in the microbial, e.g., pathogen, load indicates that the microbial species is not susceptible to the antimicrobial. In some embodiments, if the subject is receiving treatment, e.g., with an antimicrobial, a decrease in the microbial, e.g., pathogen, load indicates that the microbial species is susceptible to the antimicrobial. The specific response with regards to microbial load is dependant on the compound—host—microbe relationship. In some embodiments, the second time point is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours after the first time point.

In some embodiments, measuring microbial load can be used to measure the susceptibility of microbial species to therapeutic agents, e.g., antimicrobials, ex-vivo. In some embodiments, a sample is acquired, e.g., obtained, from a subject as described herein. In some embodiments, the microbial load is measured in a sample, and the microbial load is then measured at a second time point in the same sample, after exposure to an antimicrobial.

In some embodiments, the sample can be divided into multiple samples, e.g., aliquots. In some embodiments, the sample is divided into 1, 2, 3, 4, 5, 6, or more aliquots. In some embodiments, the sample is divided into multiple aliquots and the microbial load is measured in an untreated sample. In some embodiments, the sample is divided into multiple aliquots and one or more aliquots are treated with antimicrobials, after which the microbial load is measured.

In some embodiments, the microbial load in a sample treated with an antimicrobial is measured 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 1 hour 10 minutes, 1 hour 20 minutes, 1 hour 30 minutes, 2 hours, 2 hours 30 minutes, 3 hours, 4 hours, hours, 6 hours, or 7 hours, after treatment with the antimicrobial.

The microbial load of a sample treated with an antimicrobial can be compared with the microbial load of the same sample pre-treatment or with a different sample from the same source pre-treatment or untreated to assess the effect of the antimicrobial on the microbial species. In some embodiments, a decrease in microbial load after exposure to the antimicrobial load indicates that the microbial species is susceptible to the antimicrobial. In some embodiments, an increase in the microbial load, or no change in the microbial load, after exposure to the antimicrobial indicates that the microbial species is not susceptible, or is resistant, to the antimicrobial.

Antimicrobials include, for example, ampicillin, amoxycillin, aureomicin, bacitracin, ceftazidime, ceftriaxone, cefotaxime, cephachlor, cephalexin, cephradine, ciprofloxacin, clavulanic acid, cloxacillin, dicloxacillan, doxycycline, erythromycin, flucloxacillan, gentamicin, gramicidin, methicillan, neomycin, oxacillan, penicillin, vancomycin, capsofungin, flucytosine, fluconazole, itraconazole, ketoconazole, and miconazole.

In some embodiments, the antimicrobial is an antibiotic. In some embodiments, the antibiotic may be a compound relating to the following antibiotic classes: penicillins, tetracyclines, cephalosporins, quinolones, lincomycins, macroslides, sulfomides, glycopeptides, aminoglycosides, and/or carapenems. In some embodiements, the antibiotic may be from an alternative class of antibitioics.

In some embodiments, the antimicrobial is an antifungal. In some embodiments, the antifungal may be a compound relating to the following antifungal classes from azoles, allylamines, echinocandins, nucleoside analogs, and/or polyenes. In some embodiements, the antifungal selected may be slected from an alternative class of antifungals.

In some embodiments, the amount, concentration, or number of microorganisms present in the initial sample is determined through a calibration process. This is in contrast to methods which require culturing, and other molecular methods with a non-integrated approach.

In some embodiments, the calibration process comprises one or more calibration steps. In some embodiments, calibration for quantitative or semi-quantitative load assessment for a given load input range (i.e. 1 CFU/100 ml-100 CFU/ml) comprises comparing the results of a DIANA invasion assay using the methods described herein to the results of colony counts using the same input, e.g., the same input amount or a known relative input amount. In some embodiments, calibration for the quantitative or semi-quantitative load assessment for a given load input range comprises inputting predetermined quantities of cells. In some embodiments, calibration for the quantitative or semi-quantitative load assessment may be accomplished for a given load input range comprises inputting predetermined quantities of gDNA.

In some embodiments, quantitation or semi-quantitative is accurate within a particular input load dynamic range, e.g., between 1 and 100 to 3,000, between 2 and 100 to 3,000, between 3 and 100 to 3,000, between 4 and 100 to 3,000, between 5 and 100 to 3,000, between 6 and 100 to 3,000, between 7 and 100 to 3,000, between 8 and 100 to 3,000, between 9 and 100 to 3,000, between 10 and 100 to 3,000, between 11 and 100 to 3,000, between 12 and 100 to 3,000, between 13 and 100 to 3,000, between 14 and 100 to 3,000, between 15 and 100 to 3,000, between 16 and 100 to 3,000, between 17 and 100 to 3,000, between 18 and 100 to 3,000, between 19 and 100 to 3,000, between 20 and 100 to 3,000, between 21 and 100 to 3,000, between 22 and 100 to 3,000, between 23 and 100 to 3,000, between 24 and 100 to 3,000, between 25 and 100 to 3,000, between 26 and 100 to 3,000, between 27 and 100 to 3,000, between 28 and 100 to 3,000, between 29 and 100 to 3,000, or between 30 and 100 to 3,000 CFU or cells input. In some embodiments, the output or signal dynamic range is between about 10× and 50×, between about 20× and 100×, between about and 300×, between about 40× and 400×, between about 50× and 500×, between about 60× and 600×, between about 70× and 700×, between about 80× and 800×, between about 90× and 900×, between about 100× and 1000×, between about 100× and 1250×, between about 100 and 1500×, between about 100 and 1750×, or between about 100× and 2000×.

In some embodiments, the input load dynamic range is adjusted by varying the input volume and/or increasing or decreasing the output or yield of the enzymatic amplification step. By way of example, but not by way of limitation, should an input of 1-100 CFU (or cells), with a recalibrated optimal number of PCR cycles under the current conditions be 30, assuming a PCR cycle efficiency of 85%, a similar dynamic range of 100× could be achieved for an input of 250-2,500 CFU (or cells) by using roughly 20-22 PCR cycles.

In some embodiments, the output or yield of the enzymatic amplification step is increased or decreased to accommodate fewer or more DIANA probes in the detection step.

In some embodiments, one calibration for load assessment is performed for all organisms to be tested. In some embodiments, one calibration for load assessment is performed for all Gram-positive microorganisms to be tested. In some embodiments, one calibration for load assessment is performed for all Gram-negative microorganisms to be tested. In some embodiments, one calibration for load assessment is performed for all fungi to be tested. In some embodiments, one calibration for load assessment is performed for each genus to be tested. In some embodiments, a calibration for quantitative load assessment is performed for each organism to be quantified.

In some embodiments, separate calibrations for quantitative load assessment are done for samples having compounds that may affect the readout of the assay, e.g., antibiotics, anticoagulants, drug compounds, etc.

In some embodiments, calibration for quantitative or semi-quantitative load assessment may yield a results range. By way of example, without limitation, a given input load may yield a signal of 100±9.

In some embodiments, there may be one or more mathematical relationships between load input and signal output, for example linear, polynomial, exponential, etc.

In some embodiments, more than one microbial species will be measured and calibration for load assessment will take into account one or more of the following factors: relative lysis yields, relative amplification yields, genomic copies of the target region for amplification, DIANA capture/detection efficiency. In some embodiments, none of these factors are taken into account. In some embodiments, a subset of these factors are taken into account. In some embodiments, all of these factors are taken into account. A non-limiting example would be a case where two pathogens are present in a sample, for example two Gram-negative bacterial species. Given the ease with which these bacteria are lysed, and the single primer pair used to amplify both species, it is likely that only target genomic copies and DIANA capture/detection efficiency need to be accounted for.

In some embodiments, the ability to determine change in pathogen load, may be of use in multiple applications, by way of example but not by way of limitation, during drug/compound development processes, enrichment of clinical trials, monitoring performance of a treatment in-vitro, monitoring performance of a treatment in-vivo, determining if to alter treatment or care, establishing compound-pathogen-host relationships.

Kits

The present disclosure also provides kits for use of the DIANAs as described herein in the methods described herein. In some embodiments, the kit comprises reagents and protocols for detecting and/or identifying and/or evaluating one or more microorganisms from a sample without prior enrichment. In some embodiments, this kit contains reagents and protocols for the following processes:

(i) providing a biological sample;

(ii) lysing the mammalian cells in the sample, including those which contain DNA;

(iii) isolating a plurality of microbial genetic materials from sample;

(iv) amplifying the plurality of microbial genetic materials; and

(v) detecting, and/or identifying, and/or characterizing the microbial genetic materials, e.g., contacting the amplified microbial genetic materials with a plurality of DNA Invading Artificial Nucleic Acids (DIANAs) and detecting binding of one or more of the plurality of DIANAs to the microbial genetic material.

In some embodiments, the kit can additionally comprise instructions for use in any of the methods described herein. The included instructions may comprise a description of detecting microbial genetic material, e.g., by depleting eukaryotic DNA from a sample, lysing microbial cells, isolating genetic material, amplifying the genetic material, contacting the amplified genetic material with DIANAs, and detecting the binding. The kit may further comprise a description of obtaining a sample from a subject. In some embodiments, the instructions comprise selecting a subject for testing based on diagnostic criteria.

In some embodiments, the kit contains pre-calibrated reagents for load assessment, microbial spectrum analysis, and microbial detection.

In some embodiments, reagents are provided in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, and the like.

In some embodiments, the kit may be utilized manually (human operation). In some embodiments, usage of the kit may be automated. Non-limiting examples for automating include robotic pipetting stations, and the fluidic devices described herein.

EXAMPLES Example 1: Isolation and Amplification of Borrelia Microbial DNA from Human Whole Blood

Described herein are assays for the isolation and amplification of microbial DNA from human whole blood. DNA extraction from blood involves 6 steps: (1) lysis of eukaryotic cells; (2) Human DNA Capture; (3) Borrelia lysis; (4) target DNA capture; (5) wash; and (6) elution. Each step is described below for each volume.

(1) Mild Lysis of eukaryotic cells, leaving microbial DNA intact:

Depending on sample volume add the appropriate amounts of the following:

-   -   Magnesium Chloride     -   DI Water     -   L-Arginine     -   Fos-Choline.         Then mix samples on a shaker for 10 minutes, pass samples         through a 20 μm mesh and return samples to the shaker for 10         minutes.

(2) Human DNA Capture

Depending on sample volume add 50% of the appropriate amounts of MERPs. Incubate for 1-2 minutes and add the remaining 50% of MERPs. Incubate for 5-10 minutes to ensure complete capture of free genomic material. Place tube on magnet and immobilize MERPs. Remove supernatant and place in a fresh tube. Do not discard supernatant as it contains the microorganisms.

(3) Target Lysis

Depending on sample volume add the appropriate amounts of the following:

-   -   Triton X-100.     -   TRIS-HCl to adjust pH.     -   Sodium Chloride to adjust electrolyte strength.         Incubate samples at room temperature for 10 minutes.

(4) Target DNA Capture

Depending on sample volume add the appropriate amounts of MERPs sufficient to capture the extracted microbial DNA. After 10 minutes on the shaker, transfer tubes to a magnet rack for 8 minutes. After 8 minutes on the magnet, remove and discard supernatant.

(5) Wash

Resuspend MERPS in appropriate wash buffer. Magnetize tubes for 1 minute, remove supernatant, and repeat process 3-5 times. Rule of thumb is 2 washes after all pink/red hue is eliminated from MERP solution.

(6) Elution

Magnetize tubes for 1 minute, remove supernatant. Resuspend beads in 32 μl, of elution buffer. Then incubate samples for 5 minutes at room temperature. Then magnetize tubes for 1 minute and transfer supernatant to a 200 μl, PCR strip tube.

The microbial amplification reaction can then be carried out. Exemplary PCR amplification reagents and protocols are shown in Tables 36 and 37.

TABLE 36 PCR Master Mix Stock Final Volume Per Materials to be used: Concentration: Concentration: reaction (μL) V7 buffer 5X 1X   12 dNTPs 10X  0.2X 1.2 primer Mix -PC 22.5x 0.1X 0.3 DMSO 100% (v/v) 5% (v/v) 3 Q5 2X  0.03X 0.9 Magnesium Chloride  100 mM  2 mM 1.2 Sodium Chloride 1000 mM 20 mM 1.2 water 5.3

TABLE 37 Q_40_63 Temp (° C.) Time (s) Cycles Initial denat. 93 30 1 Denat. 93 10 4 Anneal 63 30 Extend 72 60 Denat. 92 10 36 Anneal/Extend 66/72 20/45 Final Extend 72 120 1 Soak 20 240 1

Example 2: Isolation and Amplification of Borrelia Microbial DNA from Human Whole Blood

Described herein is an invasion assay for detecting microorganisms, e.g., after isolation and amplification of microbial genetic material according to the protocol described in Example 1. The invasion mix is prepared according to Table 38 below. γPNA should be added to individual reactions rather than to the invasion mix:

TABLE 38 Number of reactions (n): Volume Per Materials to be used: reaction (μL) (n * 1.2) * 100 μL 2X Invasion Buffer 50 μL 50 * (n * 1.2)  Deionized Water (32) μL 32 * (n * 1.2)  Borrelia - LPC 2 μL 2 * (n * 1.2) γPNA probes for 2 μL 2 * (n * 1.2) Borrelia panel PCR product from (4) μL 4 * (n * 1.2) extracted sample Container(s): 5 mL tube 5 mL tube

Once the invasion mix is prepared, 98 μl, of invasion mix per reaction is transferred PCR tube along with 2 μl, of the required γPNA probe. Then begin invasion by incubating reactions at 85° C. for 7 minutes. After 7 minutes, transfer tubes to 75° C. and incubate at 75° C. for 2 minutes.

Then prepare PreWash Solution according to Table 39 below:

TABLE 39 Number of reactions (n): Volume Per Materials to be used: reaction (μL) (n * 1.2) * 100 μL 2X Invasion Buffer 50 μL   50 * (n * 1.2) Deionized Water 36.75 μL 36.75 * (n * 1.2) Sodium Chloride 6.25 μL  6.25 * (n * 1.2) Streptavidin beads 1 μL    1 * (n * 1.2) Container(s): 5 mL tube 5 mL tube

Transfer 100 μL of PreWash solution to each reaction and mix. Then incubate the reaction at 75° C. for 2 more minutes. Then move the tubes to RT for 10 minutes. Then place PCR tubes on magnet for at least 1 minute. Then remove supernatant without disturbing magnetized beads. Remove PCR tubes from magnet and resuspend beads in 10 mM NaPi, 200 μL per reaction. Then place PCR tubes back on magnet for at least 1 minute.

Prepare Antibody Solution according to Table 40 below:

TABLE 40 Number of reactions (n): Volume Per Materials to be used: reaction (μL) (n* 1.2) * 50 μL Peroxidase-Conjugated 1   1 * (n * 1.2) IgG Fraction Anti-Digoxin Blocking Buffer 16.5 16.5 * (n * 1.2) Sodium Phosphate Buffer, 32.5 32.5 * (n * 1.2) pH 7.2, 10 mM Remove supernatant without disturbing magnetized beads. Then remove PCR tubes from magnet and resuspend beads in Antibody Solution, 50 μL per reaction. Begin the antibody binding step by incubating tubes on bench at room temperature for 5-10 minutes. Once antibody binding is complete, add 150 μL of 10 mM NaPi with 0.05% Tween-20 to each tube. Then Place PCR tubes back on magnet for at least 1 minute. Remove supernatant without disturbing magnetized beads. Wash beads in 200 μL of 10 mM NaPi with 0.05% Tween-20 a total of 3 times. Samples should be transferred to a new PCR tube after 1 wash. Then place PCR tubes back on magnet for at least 1 minute. During this magnetization step, prepare Luminol Mix according to Table 41 below:

TABLE 41 Number of reactions (n): Volume Per Materials to be used: reaction (μL) (n * 1.2) * 50 μL Luminol Enhancer 25 25 * (n * 1.2) Peroxide Solution 25 25 * (n * 1.2)

Remove supernatant without disturbing magnetized beads. Remove PCR tubes from magnet and resuspend beads in Luminol Mix, 50 μL per reaction. Then immediately transfer resuspended beads into opaque-walled 96-well plate and read plate.

Example 3: Selective Lysis of Leukocytes and Effective Lysis of Borrelia Cells

Demonstrated herein in is that the selective lysis solution does not impact the integrity of Borrelia spirochetes. Data is shown in FIG. 10 . Viability studies were conducted in which B. burgdorferi at a concentration of 10 cells/R₁ was incubated in the selective lysis solution for 30 min (Lane 1 of FIG. 10A) and 60 min (Lane 3 of FIG. 10A), respectively. This is far longer than required for leukocyte lysis. After incubation the reaction was spun down to remove all cells and 3 μl of the supernatant was loaded into a PCR reaction amplifying the omp66. No free Borrelia DNA was detected (see gel and intensity graph), where the positive controls (Lanes 2 and 4 of FIG. 10A) yielded highly visible omp66 amplicon at the same 30 cells/reaction (i.e. 45 fg/reaction). Secondary control bands serve as an internal control to verify reaction integrity.

To ensure that the selective lysis solution effectively lyses leukocytes, cell cytometry was used to verify that >99% of leukocytes are eliminated after 5 minutes. To improve on the resolution of the cell counter, the amount of hDNA remaining after microbial DNA isolation (Step III of FIG. 2 ) was quantified. Total lysis directly correlated to the amount remaining after the preceding selective lysis. The calculated removal rate was 99.95%±0.04% of the human DNA from 20 ml whole-blood (n=24), a value experimentally determined to not inhibit downstream PCR processes.

It was further demonstrated that Borrelia spirochetes are readily lysed in the presence of our Total Microbial Lysis Solution (Step II of FIG. 2 ), where via cell cytometry we verified >99% elimination of spirochetes in 5 min. In addition, via PCR/omp66 assays, amplification of a purified and quantified aliquot of Borrelia gDNA was compared to an aliquot of Borrelia cells having been exposed to our Total Microbial Lysis Solution yielding similar results (see FIG. 10B). Load was defined as either 40 genomic equivalents (i.e. 60 fg DNA) or 40 cells (quantified via cytometry), lysed and purified via the disclosed processes.

Example 4: DIANA Based Species Level ID of Borrelia Species

Species level identification of Borrelia using DIANAs, γPNAs in this case, is shown in FIG. 11 . This is done through the use of an example Borrelia test menu encompassing γPNA detection probes for: (1) Broad Borrelia, (2) B. burgdoferi, (3) B. afzelii, (4) B. garinii, and (5) B. mayonii. In each study, roughly 2.5×10⁻¹⁵ moles of amplicon derived from the omp66 gene was used. Note that in each study (n=3) only the correct detection probe yielded a discernable signal where the typical signal to off-target ratio was >500:1.

Example 5: Ultra-Sensitive Detection of Borrelia Directly from Blood

The suitability of ultrasensitive detection methods described in Examples 1 and 2 (i.e., RaPID) for the direct molecular detection of Borrelia from whole-blood is shown in FIG. 12 . Data is presented for both single cell/ml and sub-cell/ml loads spiked directly into fresh human whole-blood. In these studies, differing cell loads (B. burgdorferi were spiked directly into a 20 ml blood (n=3 each) and processed as a single reaction. All results yielded a clearly distinguishable signal roughly 100-600× above the cut-off defined as 3× std.dev above the mean NIC (Non-Infected Control) signal (n=20).

Using the same processes and test menu discussed in Example 4 encompassing (1) Broad Borrelia, (2) B. burgdoferi, (3) B. afzelii, (4) B. garinii, and (5) B. mayonii, these capabilities were likewise demonstrated for a clinically viable test menu in FIG. 13 .

Example 6: Ultra-Sensitive Detection of Bacteria Directly from Blood

Data using was generated using a model pathogen (E. faecium) for ultra-sensitive detection of cells using the methods described in Examples 1 and 2 (i.e., RaPID). The results are presented in FIG. 14 . Data demonstrates an increase in signal of 5-10× when transitioning from 1.5 ml to 10 ml whole-blood (in line with the theoretical 6-7× signal increase), and an ability to reach sub-1 CFU/ml loads, at the 10 ml input volume. These results demonstrate both the value of assaying larger volumes of blood and our ability to detect ultra-low loads.

EQUIVALENTS

The foregoing written specification is considered to be sufficient to enable one ordinarily skilled in the art to practice the invention. The present invention is not to be limited in scope by examples provided, since the examples are intended as mere illustrations of one or more aspects of the invention. Other functionally equivalent embodiments are considered within the scope of the invention. Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Each of the limitations of the invention can encompass various embodiments of the invention. It is, therefore, anticipated that each of the limitations of the invention involving any one element or combinations of elements can be included in each aspect of the invention. This invention is not limited in its application to the details of construction and the arrangement of components set forth or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways.

Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including,” “comprising,” or “having,” “containing”, “involving”, and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.

All references, patents and patent applications that are recited in this application are incorporated by reference herein in their entirety. 

1. A composition consisting essentially of: a magnesium salt; a buffering agent; water; and a compound of Formula 1:

wherein R₁ is selected from the group consisting of optionally substituted, branched or unbranched, saturated or unsaturated C₁-C₈ aliphatic; optionally substituted, saturated or unsaturated C₃-C₁₄ carbocyclic; optionally substituted, saturated or unsaturated 3-8 membered heterocyclic; optionally substituted, branched or unbranched, saturated or unsaturated ((R_(a))_(q)—(C═O)—(R_(a))_(q))_(p); optionally substituted C₆-C₁₄ aryl; and optionally substituted 3-8 membered heteroaryl; and/or any suitable combinations thereof; wherein R₂ is selected from the group consisting of hydrogen; optionally substituted, branched or unbranched, saturated or unsaturated C₁-C₂₈ aliphatic; optionally substituted, branched or unbranched, saturated or unsaturated —(R_(b)—(O—R_(b))_(n)—O—R_(b))_(p); optionally substituted, branched or unbranched, saturated or unsaturated —(R_(b)—(O—R_(b))_(n)—NH—R_(b))_(p); optionally substituted, branched or unbranched, saturated or unsaturated —(R_(b)—(O—R_(b)—O)_(n)—S—R_(b))_(p); optionally substituted, branched or unbranched, saturated or unsaturated —(R_(b)—(S—R_(b))_(n)—S—R_(b))_(p); optionally substituted C₆-C₁₄ aryl; optionally substituted 3-8 membered heteroaryl; optionally substituted, saturated or unsaturated C₃-C₁₄ carbocyclic; optionally substituted, saturated or unsaturated 3-8 membered heterocyclic; optionally substituted, branched or unbranched, saturated or unsaturated —(C═O)—(R_(b)); optionally substituted, branched or unbranched, saturated or unsaturated —((R_(a))_(q)—O—(R_(a))_(q))_(p)—; optionally substituted, branched or unbranched, saturated or unsaturated —((R_(a))_(q)—NH—(R_(a))_(q))_(p)—; optionally substituted, branched or unbranched, saturated or unsaturated —((R_(a))_(q)—N(R_(a))—(R_(a))_(q))_(p)—; and optionally substituted, branched or unbranched, saturated or unsaturated —((R_(a))_(q)—S—(R_(a))_(q))_(p)—; and/or any suitable combinations thereof; wherein each occurrence of R a is independently C₁-C₈ aliphatic or C₆-C₁₄ aryl; wherein each occurrence of R_(b) is independently C₁-C₁₅ aliphatic or C₆-C₁₄ aryl; wherein each occurrence of subscript q is independently an integer between 0 and 1, inclusive; wherein each occurrence of subscript p is independently an integer between 1 and 6, inclusive; and wherein each occurrence of subscript n is independently an integer between 0 and 14, inclusive.
 2. The composition of claim 1, further comprising blood.
 3. The composition of claim 2, wherein the composition comprises between 20% and 60%, inclusive, of the blood by volume.
 4. The composition of claim 2, wherein the blood comprises microbial cells.
 5. A kit, comprising: the composition of claim 1; and an anion-exchanger.
 6. The kit of claim 5, wherein the kit further comprises microparticles.
 7. The kit of claim 5, wherein the anion-exchanger comprises microparticles.
 8. The kit of claim 5, wherein the anion-exchanger comprises magnetizable, electro-reactive microparticles.
 9. The kit of claim 5, further comprising a composition comprising cell lysis reagents that allow for the lysis of microbial cells.
 10. The kit of claim 5, further comprising a fluidic device.
 11. The kit of claim 5, wherein the buffering agent comprises a polar small molecule and/or an inorganic salt.
 12. The kit of claim 5, wherein the magnesium salt comprises an inorganic counter ion.
 13. The kit of claim 5, wherein the magnesium salt is selected from the group consisting of MgCl₂, MgCO₃, MgSO₄, and MgBr₂.
 14. The kit of claim 5, wherein R₂ is selected from the group consisting of optionally substituted, branched or unbranched, saturated or unsaturated C₁-C₂₈ aliphatic; optionally substituted, branched or unbranched, saturated or unsaturated —(R_(b)—(O—R_(b))_(n)—O—R_(b))_(p); optionally substituted, branched or unbranched, saturated or unsaturated —(R_(b)—(O—R_(b))_(n)—NH—R_(b))_(p); optionally substituted, branched or unbranched, saturated or unsaturated —(R_(b)—(O—R_(b)—O)_(n)—S—R_(b))_(p); optionally substituted, branched or unbranched, saturated or unsaturated —(R_(b)—(S—R_(b))_(n)—S—R_(b))_(p); optionally substituted C₆-C₁₄ aryl; optionally substituted 3-8 membered heteroaryl; optionally substituted, saturated or unsaturated C₃-C₁₄ carbocyclic; optionally substituted, saturated or unsaturated 3-8 membered heterocyclic; optionally substituted, branched or unbranched, saturated or unsaturated —(C═O)—(R_(b)); optionally substituted, branched or unbranched, saturated or unsaturated —((R_(a))_(q)—O—(R_(a))_(q))_(p)—; optionally substituted, branched or unbranched, saturated or unsaturated —((R_(a))_(q)—NH—(R_(a))_(q))_(p)—; optionally substituted, branched or unbranched, saturated or unsaturated —((R_(a))_(q)—N(R_(a))—(R_(a))_(q))_(p)—; and optionally substituted, branched or unbranched, saturated or unsaturated —((R_(a))_(q)—S—(R_(a))_(q))_(p)—; and/or any suitable combinations thereof; wherein each occurrence of R a is independently C₁-C₈ aliphatic or C₆-C₁₄ aryl; wherein each occurrence of R_(b) is independently C₁-C₁₅ aliphatic or C₆-C₁₄ aryl; wherein each occurrence of subscript q is independently an integer between 0 and 1, inclusive; wherein each occurrence of subscript p is independently an integer between 1 and 6, inclusive; and wherein each occurrence of subscript n is independently an integer between 0 and 14, inclusive.
 15. The kit of claim 5, wherein R₁ is independently selected from the group consisting of optionally substituted, branched or unbranched C₁-C₈ alkyl; optionally substituted, branched or unbranched C₂-C₈ alkenyl; and optionally substituted, branched or unbranched C₂-C₈ alkynyl.
 16. The kit of claim 5, wherein R₁ is optionally substituted, branched or unbranched C₁-C₈ alkyl.
 17. The kit of claim 5, wherein R₂ is independently selected from the group consisting of optionally substituted, branched or unbranched C₁-C₂₈ alkyl and optionally substituted, branched or unbranched C₂-C₂₈ alkenyl.
 18. The kit of claim 5, wherein the compound of Formula 1 is selected from the group consisting of:


29. The kit of claim 5, wherein the compound of Formula 1 is


20. The kit of claim 5, wherein the pH has a value of between 8 and 11.5, inclusive. 